Identification of receptor and heparin binding sites in FGF4 by structure-based mutagenesis

ABSTRACT

The present invention provides methods and compositions that may be used to modulate binding of a fibroblast growth factor (FGF) polypeptide to an FGF receptor (FGFR). In preferred embodiments, the methods and compositions of the invention modulate binding of a particular FGF polypeptide, the FGF4 polypeptide, to its receptor. The invention provides, in particular, variant FGF polypeptides that have at least one amino acid residue substitutions, insertion or deletion which alters the polypeptdies&#39; binding affinity for an FGFR. The invention also provides models for the three-dimensional structure of a dimerized complex of FGF-FGFR-heparin. Using these models, key amino acid residues are identified and novel compounds (including novel variants of FGF and FGFR) can be identified which modulate FGF binding to its receptor. Such new compounds are therefore useful, e.g., as agonist and antagonist for FGF signaling and for bioactivities associated therewith.

This application is a continuation of PCT/US02/24274 filed Aug. 1, 2002and claims priority under 35 U.S.C. 119 (e) from Provisional ApplicationNo. 60/309,431, filed Aug. 1, 2001, incorporated herein by reference inits entirety.

This invention as made with Government support under Grant Nos. DE13686and CA42568 awarded by the National Institutes of Health. The UnitedStates Government may have certain rights to this invention pursuant tothe terms of those grants.

1. FIELD OF THE INVENTION

The present invention relates to a class of proteins known as fibroblastgrowth factor (FGF) proteins or FGF ligands. The invention also relatesto receptors, known as fibroblast growth factor receptors (FGFRs), thatrecognize and specifically bind to FGF proteins. The invention furtherrelates to a particular FGF polypeptide, referred to as FGF4. Themethods and compositions of the invention relate to novel methods andcompositions, including novel polypeptides and other compounds, thatmodulate FGF binding to its receptor and may be used, e.g., as agonistsor antagonists to modulate FGF receptor activity and/or biologicalactivities that are associated with FGF.

2. BACKGROUND OF THE INVENTION

The fibroblast growth factor (FGF) family of proteins comprises at least22 polypeptides, referred to as FGF1-FGF22, with diverse biologicalactivities. For reviews, see, McKeehan et al., Prog. Nucleic Acid Res.Mol. Biol. 1998, 59:135-176; Nishimura et al., Biochim Biophys. Acta.2000, 1492:203-206; Yamashita et al., Biochem. Biophys. Res. Commun.2000, 277:494-498. For example, FGF polypeptides modulate theproliferation and differentiation of a variety of cells of mesenchymaland neuro-ectodermal origin (Basilico & Moscatelli, Adv. Cancer Res.1992, 59:115-165). FGF polypeptides also play critical roles duringembryonic processes such as mesoderm induction, post-implantationblastocyst development, and limb and lung development (Goldfarb,Cytokine Growth Factor Rev. 1996, 7:311-325; Xu et al., Cell Tissue Res.1999, 296:33-43). Increased FGF signaling leads to a variety of humanskeletal disorders, including dwarfism and craniosynostosis syndromes(McIntosh et al., Cell Struct. Funct. 2000, 25:85-96; Naski & Omitz,Front. Biosci. 1998, 3:D781-D794; Wilkie, Hum. Mol. Genet. 1997,6:1647-1656). In adult organisms FGFs are thought to be involved inphysiological angiogenesis and wound healing as well as in pathologicalangiogenesis such as in tumor neovascularization and diabeticretinopathy (Basilico & Moscatelli, Adv. Cancer Res. 1992, 59:115-165).

The diverse effects of FGFs are mediated by at least four receptortyrosine kinases, which are referred to collectively as the FGF receptor(FGFR) polypeptides and are known individually as FGFR1-FGFR4. The FGFRpolypeptides comprise an extracellular domain, a single transmembranehelix and a cytoplasmic portion. The extracellular domain binds to theFGF polypeptide ligand, and may be subdivided into at least threedistinct three immunoglobulin (Ig)-like domains, known as D1-D3, witheach domain being connected by a “linker” polypeptide sequence. Ligandbinding and specificity resides in the D2 and D3 domains and the shortD2-D3 linker (Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al.,Cell 2000, 101:413-424; Stauber et al., Proc. Natl. Acad. Sci. U.S.A.2000, 97:49-54).

FGFR dimerization is prerequisite for FGF signaling and requires heparinor heparan sulfate proteoglycans (HSPG) (Ornitz, Bioessays 2000,22:108-112; Schlessinger, Cell 2000, 103:211-225). The recent crystalstructure of a ternary FGF2-FGFR1-heparin complex has provided amechanistic view of the process by which heparin aids FGF polypeptidesto induce FGFR dimerization (Schlessinger et al., Mol. Cell 2000,6:743-750). According to this “two end” model, heparin interacts via itsnon-reducing end with the heparin binding sites of FGF and FGFR andpromotes the formation of a ternary 1:1:1 FGF:FGFR:heparin complex. Asecond ternary 1:1:1 FGF:FGFR:heparin complex is then recruited to thefirst ternary complex via interactions of FGFR, FGF and heparin in oneternary complex with the FGFR in the adjoining ternary complex.

A fundamentally different model for FGFR dimerization has emerged fromthe recent crystal structure of a dimeric FGF1-FGFR2-heparin ternarycomplex (Pellegrini et al., Nature 2000, 407:1029-1034). In thisstructure, a single heparin molecule links two FGF ligands into a dimerthat bridges between two receptor chains. The asymmetric heparin bindinginvolves contacts with both FGF molecules but only one receptor chain.There is essentially no protein-protein interface between the two 1:1FGF-FGFR complexes in the dimer.

With the exception of FGF1, which is the universal ligand for all FGFRs,most FGF polypeptides exhibit specific, albeit promiscuous, patterns ofreceptor binding affinity (Ornitz et al., J. Biol. Chem. 1996,271:15292-15297). Comparison of the crystal structures of FGF1-FGFR1,FGF2-FGFR1 and FGF2-FGFR2 complexes defined a general binding interfacefor FGF-FGFR complexes involving contacts made by FGF to D2 and to theD2-D3 linker (Plotnikov et al., Cell 2000, 101:413-424). It has alsobeen shown that specificity is achieved through interactions of the FGFN-terminal (i.e., the amino acid sequence immediately preceding the FGFpolypeptide's β-trefoil core domain) and central regions with FGFR D3.These structures have also provided a molecular basis for howalternative splicing in FGFR modulates specificity. In both FGF2-FGFR1and FGF2-FGFR2 structures, FGF2 makes specific contacts with the βC′-βEloop in D3, which is subject to alternative splicing. Consequently, FGF2discriminates between the IIIc and IIIb variants of FGFRs. In contrast,FGF1 does not interact with the βC′-βE loop and therefore can bind allFGFRs irrespective of alternative splicing in D3 (Plotnikov et al., Cell2000, 101:413-424).

FGF4 shares about 30% sequence identity with the prototypical members ofthe FGF family, FGF1 and FGF2 (Delli Bovi et al., Cell 1987,50:729-737). FGF4, unlike FGF1 and FGF2, has a classical signal peptidpand thus is efficiently secreted from cells (Bellosta et al., J. CellBiol. 1993, 121:705-713). Most receptor binding studies indicate thatFGF4 binds and activates the IIIc splice forms of FGFR1-3 to comparablelevels, but it shows little activity towards the IIIb splice forms ofFGFR1-3 as well as towards FGFR4 (Ornitz et al., J. Biol. Chem. 1996,271:15292-15297; Vainikka et al., EMBO J. 1993, 11:4273-4280). As forFGF1 and FGF2, heparin greatly augments the biological activity of FGF4on cells lacking endogenous cell surface HSPG (Mansukhani et al., Proc.Natl. Acad. Sci. U.S.A. 1992, 89:3305-3309). However, employingselectively O-desulfated heparins, Guimond et al. (J. Biol. Chem. 1993,268:23906-23914) have shown that both 2-O-and 6-O-desulfated heparinwere able to support the mitogenic activity of FGF4, while neither ofthese heparins could support the biological activity of FGF1 and FGF2.It has therefore been suggested the sulfation motifs in heparin requiredfor FGF4 activity may differ from those required for FGF1 and FGF2actions (Guimond et al., supra; Ishihara, Glycobiology 1994, 4:817-824).

In summary, the exact interactions that stabilize complexes of the FGF4polypeptide with its receptor and/or heparin remain poorly understood.Yet, given the range of biological disorders associated with FGFsignaling, there is an urgent need to identify and characterize theseinteractions. There exist, moreover, a need to identify compounds thatmodulate binding of FGF4 to either an FGF receptor or heparin, includingmutant or variant forms of the FGF4 polypeptide that have alteredbinding affinities, as well as other compounds that may be agonists orantagonists of FGF4 binding and/or activity.

3. SUMMARY OF THE INVENTION

The present invention provides mutant FGF4 polypeptides, wherein atleast one amino acid residue in the primary binding site, the secondarybinding site or the heparin binding site is different from the wild-typeFGF4 molecule.

In preferred embodiments, the invention provides the following mutantFGF4 polypeptides, each containing the substitution at the indicatedresidue with alanine: tyrosine at amino acid residue 87; phenylalanineat amino acid residue 129; phenylalanine at amino acid residue 151;glutamic acid at amino acid residue 159; phenylalanine at amino acidresidue 166; leucine at amino acid residue 203; arginine at amino acidresidue 205; asparagine at amino acid residue 89; lysine at amino acidresidue 198; asparagine at amino acid residue 89; lysine at amino acidresidue 183; lysine at amino acid residue 188; lysine at amino acidresidue 183; arginine at amino acid residue 103; lysine at amino acidresidue 144; and arginine at amino acid residue 103.

In another preferred embodiment, the invention provides a mutant FGF4polypeptide containing two alanine substitutions at lysine residue 144and arginine residue 103.

The invention further provides a crystal of FGF4, the crystal belongingto the orthorhombic space group P212121 and having unit cell dimensionsa=40.37, b=53.3 and c=56.23.

In a preferred embodiment, the crystal comprises an FGF4 polypeptidehaving the amino acid sequence depicted in FIG. 2 (top line).

The present invention also provides a method for producing mutant FGF4polypeptides, and methods of testing mutant FGF4 polypeptides forincreased or decreased binding and/or activity.

Crystalline forms of FGF4 are also provided by the present invention.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ribbon diagram illustrating the tertiary structure for anFGF4 polypeptide whose amino acid sequence is set forth in FIG. 2,below. The secondary structure assignments were assigned using theprogram PRODCHECK (Laskowski et al., J. Appl. Cryst. 1993, 26:283-291),and the figure created using the programs Molscript (Kraulis, J. Appl.Crystallogr. 1991, 24:946-950) and Raster3D (Merritt & Bacon, MethodsEnzymol. 1997, 277:505-524). β-strands are labeled according toconventional strand nomenclature for FGF1 and FGF2 (see, Faham, Curr.Opin. Struct. Biol. 1998, 8:578-586). NT and CT denote the amino- andcarbyoxy-termini, respectfully.

FIG. 2A-F provides a structure based sequence alignment of the FGF4polypeptide amino acid sequence (top line) whose structure isillustrated in FIG. 1A, and related human FGF polypeptides FGF6, FGF1and FGF2. The alignment was generated using the program CLUSTALW(Stauber et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97:49-54). Thelocation and length of the β-strands and α-helices of the proteins'structures are indicated on top of the alignment, and the box indicatesboundaries of the β-trefoil core region or domain. Periods in thealigned sequences indicated amino acid residues that are identical tothe amino acid residue of FGF4 at that position of the alignment,whereas dashes represent gaps introduced to optimize the alignment.Residues with the β-trefoil core region are labeled to indicate theregion of FGFR1 with which they interact: residues that interact with D2are shaded (2B), residues that interact with the FGFR1 linker region(2F), residues that interact with the D3 domain of FGFR1 (2A), and FGFRresidues that interact with the βC′-βE loop in the FGFR1 D3 domain arealso shaded (2E). Also, amino acid residues that constitute theconventional “low” and “high” affinity heparin binding sites (2C) andFGF4 amino acid residues that localize to the periphery of the “high”affinity heparin binding site (and may, therefore interact withheparin-2D) are also indicated. A star indicates those amino acidresidues that are tested in the Examples, infra, by site-directedmutagenesis.

FIG. 3 provides a representative model (generated using the Molscriptand Raster3D programs) for the three-dimensional structure of anFGF4-FGFR1 complex generated by superimposition of Cα traces within theβ-trefoil of a structure for unbound FGF4 onto corresponding Cα tracesof FGF2 in a structure for an FGF2-FGFR1 complex (see the Examples,infra). FGF4, D2 of FGFR1, D3 of FGFR1, and the D2-D3 are labeled. NTand CT denote the amino- and carboxy-termini, respectfully.

FIG. 4 shows a plot comparing the binding affinities of various FGF4mutants twoards FGFP2 Data are expressed as percent inhibition ofwild-type FGF4 binding to FGFR2 by the indicated amount of unlabeledmutant FGF4.

FIGS. 5A-B show plots illustrating the differential effect of heparin onstimulation of DNA synthesis in NIH 3T3 cells by different mutant FGF4polypeptides that are described in the Examples, infra. FIG. 5A plotsdata from a representative experiments using the E159A and L203Amutants. FIG. 5B plots data from representative experiments using theK183A/K188A and the N89A/K198A double mutants.

FIG. 6 provides a representative model (generated using the Molscriptand Raster3D programs) for a dimeric complex of FGF4-FGFR1-heparin. Themodel was created by superimposition of the Cα traces for two FGF4structures onto Cα traces of the two FGF2 molecules in a ternarystructure for FGF2-FGFR1-heparin (see, the Examples, infra). NT and CTdenote the amino- and carboxy-termini, respectively, of the FGFR1polypeptide. The FGF4, FGFR1, D2 and D3 domains are labeled. Heparinoligosaccharides are rendered in ball-and-stick.

FIG. 7A-B shows the primary binding site, i.e., the surface throughwhich FGF4 binds to FGFR in the context of the FGF4-FGFR-heparin dimer.The shaded region in the three-dimensional model (7A), and in the aminoacid sequence (7B) depicts the primary binding sites. The primarybinding sites in the amino acid sequences are also shown for FGF6 (SEQID NO:21) and FGF1 (SEQ ID NO:22) in 7B.

FIG. 8A-B shows the secondary binding site, i.e., the surface whichbinds to a second FGFR molecule in the context of the FGF4-FGFR-heparindimer. The secondary binding site is depicted as shaded in both thethree-dimensional model (8A), and the amino acid sequence (8B). Thesecondary binding sites in the amino acid sequences are also shown forFGF6 (SEQ ID NO:21) and FGF1 (SEQ ID NO:22) in 8B.

FIG. 9A-B shows the heparin binding site, i.e., the surface which bindsto heparin in the context of the FGF4-FGFR-heparin dimer. The primarybinding site is depicted as shaded in both the three-dimensional model(9A), and the amino acid sequence (9B). The heparmn binding sites in theamino acid sequences are also shown for FGF6 (SEQ ID NO:21) and FGF1(SEQ ID NO:22) in 9B.

5. DETAILED DESCRIPTION OF THE INVENTION

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

5.1 General Definitions

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, a cDNA, or a restriction fragment. Inanother embodiment, an isolated nucleic acid is preferably excised fromthe chromosome in which it may be found, and more preferably is nolonger joined to non-regulatory, non-coding regions, or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acid molecules include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like. Thus, in aspecific embodiment, a recombinant nucleic acid is an isolated nucleicacid. An isolated protein may be associated with other proteins ornucleic acids, or both, with which it associates in the cell, or withcellular membranes if it is a membrane-associated protein. An isolatedorganelle, cell, or tissue is removed from the anatomical site in whichit is found in an organism. An isolated material may be, but need notbe, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography (includingpreparative solid phase chromatography, oligonucleotide hybridization,and triple helix chromatography), ultracentrifrigation, and other means.Polypeptides and proteins can be purified by various methods including,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange andpartition chromatography, precipitation and salting-out chromatography,extraction, and countercurrent distribution. For some purposes, it ispreferable to produce the polypeptide in a recombinant system in whichthe protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a polyhistidine sequence, ora sequence that specifically binds to an antibody, such as FLAG and GST.The polypeptide can then be purified from a crude lysate of the hostcell by chromatography on an appropriate solid-phase matrix.Alternatively, antibodies produced against the protein or againstpeptides derived therefrom can be used as purification reagents. Cellscan be purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting [FACS]). Other purification methods are possible. Apurified material may contain less than about 50%, preferably less thanabout 75%, and most preferably less than about 90%, of the cellularcomponents with which it was originally associated. The “substantiallypure” indicates the highest degree of purity which can be achieved usingconventional purification techniques known in the art.

A “sample” as used herein refers to a biological material which can betested, e.g., for the presence of an FGF polypeptide or FGF nucleic acidor, alternatively, for the presence of an FGFR polypeptide or nucleicacid (e.g., to identify cells that specifically express either FGF orFGFR). Such samples can be obtained from any source, including tissue,blood and blood cells, and cell culture.

Non-human animals include, without limitation, laboratory animals suchas mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animalssuch as dogs and cats; and farm animals such as sheep, goats, pigs,horses, and cows.

In preferred embodiments, the terms “about” and “approximately” shallgenerally mean an acceptable degree of error for the quantity measuredgiven the nature or precision of the measurements. Typical, exemplarydegrees of error are within 20 percent (%), preferably within 10%, andmore preferably within 5% of a given value or range of values.Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes, for example,polypeptides and polynucleotides.

The term “therapeutically effective dose” refers to that amount of acompound or compositions that is sufficient to result in a desiredactivity.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar adverse reaction (for example, gastricupset, dizziness and the like) when administered to an individual.Preferably, and particularly where a vaccine is used in humans, the term“pharmaceutically acceptable” may mean approved by a regulatory agency(for example, the U.S. Food and Drug Administration) or listed in agenerally recognized pharmacopeia for use in animals (for example, theU.S. Pharmacopeia).

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Sterile water or aqueous salinesolutions and aqueous dextrose and glycerol solutions are preferablyemployed as carriers, particularly for injectable solutions. Exemplarysuitable pharmaceutical carriers are described in “Reminington'sPharmaceutical Sciences” by E. W. Martin.

5.2 Molecular Biology Definitions

In accordance with the present invention, there may be employedconventional molecular biology, microbiology and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, for example, Sambrook, Fitsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referred toherein as “Sambrook et al., 1989”); DNA Cloning: A Practical Approach,Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. E. Perbal, APractical Guide to Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linkedtogether. For example, a “dimer” is a compound in which two buildingblocks have been joined togther; a “trimer” is a compound in which threebuilding blocks have been joined together; etc.

The term “polynucleotide” or “nucleic acid molecule” as used hereinrefers to a polymeric molecule having a backbone that supports basescapable of hydrogen bonding to typical polynucleotides, wherein thepolymer backbone presents the bases in a manner to permit such hydrogenbonding in a specific fashion between the polymeric molecule and atypical polynucleotide (e.g., single-stranded DNA). Such bases aretypically inosine, adenosine, guanosine, cytosine, uracil and thymidine.Polymeric molecules include “double stranded” and “single stranded” DNAand RNA, as well as backbone modifications thereof (for example,methylphosphonate linkages).

Thus, a “polynucleotide” or “nucleic acid” sequence is a series ofnucleotide bases (also called “nucleotides”), generally in DNA and RNA,and means any chain of two or more nucleotides. A nucleotide sequencefrequently carries genetic information, including the information usedby cellular machinery to make proteins and enzymes. The terms includegenomic DNA, cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and antisense polynucleotides. Thisincludes single- and double-stranded molecules; i.e., DNA-DNA, DNA-RNA,and RNA-RNA hybrids as well as “protein nucleic acids” (PNA) formed byconjugating bases to an amino acid backbone. This also includes nucleicacids containing modified bases, for example, thio-uracil, thio-guanineand fluoro-uracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.). Polynucleotides may contain one or more additional covalentlylinked moieties, such as proteins (e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine,psoralen, etc.), chelators (e.g., metals, radioactive metals, iron,oxidative metals, etc.) and alkylators to name a few. Thepolynucleotides may be derivatized by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidite linkage. Furthermore, thepolynucleotides herein may also be modified with a label capable ofproviding a detectable signal, either directly or indirectly. Exemplarylabels include radioisotopes, fluorescent molecules, biotin and thelike. Other non-limiting examples of modification which may be made areprovided, below, in the description of the present invention.

A “polypeptide” is a chain of chemical building blocks called aminoacids that are linked together by chemical bonds called “peptide bonds”.The term “protein” refers to polypeptides that contain the amino acidresidues encoded by a gene or by a nucleic acid molecule (e.g., an mRNAor a cDNA) transcribed from that gene either directly or indirectly.Optionally, a protein may lack certain amino acid residues that areencoded by a gene or by an mRNA. For example, a gene or mRNA moleculemay encode a sequence of amino acid residues on the N-terminus of aprotein (i.e., a signal sequence) that is cleaved from, and thereforemay not be part of, the final protein. A protein or polypeptide,including an enzyme, may be a “native” or “wild-type”, meaning that itoccurs in nature; or it may be a “mutant”, “variant” or “modified”,meaning that it has been made, altered, derived, or is in some waydifferent or changed from a native protein or from another mutant. SeeAppendix A for the three letter and one letter abbreviations for 20amino acids.

A “ligand” is, broadly speaking, any molecule that binds to anothermolecule. In preferred embodiments, the ligand is either a solublemolecule or the smaller of the two molecules or both. The other moleculeis referred to as a “receptor”. In preferred embodiments, both a ligandand its receptor are molecules (preferably proteins or polypeptides)produced by cells. Preferably, a ligand is a soluble molecule and thereceptor is an integral membrane protein (i.e., a protein expressed onthe surface of a cell). In a particularly preferred embodiment of theinvention the ligand is a fibroblast growth factor (FGF) and thereceptor is a fibroblast growth factor receptor (FGFR).

The binding of a ligand to its receptor is frequently a step of signaltransduction with a cell. For example, in preferred embodiments where aligand is an FGF polypeptide and a receptor is an FGFR polypeptide, thebinding of FGF to the FGFR polypeptide may lead to activation of atyrosine kinase activity within the FGFR polypeptide. Activation of thetyrosine kinase activity may, in turn, initiate other activitiesassociated with FGF signaling, including but not limited to mitogenesisand angiogensis. Other exemplary ligand-receptor interactions include,but are not limited to, binding of a hormone to a hormone receptor (forexample, the binding of estrogen to the estrogen receptor) and thebinding of a neurotransmitter to a receptor on the surface of a neuron.

“Amplification” of a polynucleotide, as used herein, denotes the use ofpolymerase chain reaction (PCR) to increase the concentration of aparticular DNA sequence within a mixture of DNA sequences. For adescription of PCR see Saiki et al., Science 1988, 239:487.

“Chemical sequencing” of DNA denotes methods such as that of Maxam andGilbert (Maxam-Gilbert sequencing; see Maxam & Gilbert, Proc. Natl.Acad. Sci. U.S.A. 1977, 74:560), in which DNA is cleaved usingindividual base-specific reactions.

“Enzymatic sequencing” of DNA denotes methods such as that of Sanger(Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 1977, 74:5463) andvariations thereof well known in the art, in a single-stranded DNA iscopied and randomly terminated using DNA polymerase.

A “gene” is a sequence of nucleotides which code for a functional “geneproduct”. Generally, a gene product is a functional protein. However, agene product can also be another type of molecule in a cell, such as anRNA (e.g., a tRNA or a rRNA). For the purposes of the present invention,a gene product also refers to an mRNA sequence which may be found in acell. For example, measuring gene expression levels according to theinvention may correspond to measuring mRNA levels. A gene may alsocomprise regulatory (i.e., non-coding) sequences as well as codingsequences. Exemplary regulatory sequences include promoter sequences,which determine, for example, the conditions under which the gene isexpressed. The transcribed region of the gene may also includeuntranslated regions including introns, a 5′-untranslated region(5′-UTR) and a 3′-untranslated region (3′-UTR).

A “coding sequence” or a sequence “encoding” an expression product, suchas an RNA, polypeptide, protein or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein or enzyme; i.e., the nucleotide sequence “encodes”that RNA or it encodes the amino acid sequence for that polypeptide,protein or enzyme.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlyfound, for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control of” or is “operativelyassociated with” transcriptional and translational control sequences ina cell when RNA polymerase transcribes the coding sequence into RNA,which is then trans-RNA spliced (if it contains introns) and, if thesequence encodes a protein, is translated into that protein.

The term “express” and “expression” means allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing RNA (such as rRNA or mRNA) or a protein by activating thecellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed by acell to form an “expression product” such as an RNA (e.g., a mRNA or arRNA) or a protein. The expression product itself, e.g., the resultingRNA or protein, may also be said to be “expressed” by the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a eukaryotic cell. The term “transformation” means the introductionof a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNAsequence into a prokaryotic host cell so that the host cell will expressthe introduced gene or sequence to produce a desired substance, in thisinvention typically an RNA coded by the introduced gene or sequence, butalso a protein or an enzyme coded by the introduced gene or sequence.The introduced gene or sequence may also be called a “cloned” or“foreign” gene or sequence, may include regulatory or control sequences(e.g., start, stop, promoter, signal, secretion or other sequences usedby a cell's genetic machinery). The gene or sequence may includenonfunctional sequences or sequences with no known function. A host cellthat receives and expresses introduced DNA or RNA has been “transformed”and is a “transformant” or a “clone”. The DNA or RNA introduced to ahost cell can come from any source, including cells of the same genus orspecies as the host cell or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors may include plasmids, phages, viruses, etc. and arediscussed in greater detail below.

A “cassette” refers to a DNA coding sequence or segment of DNA thatcodes for an expression product that can be inserted into a vector atdefined restriction sites. The cassette restriction sites are designedto ensure insertion of the cassette in the proper reading frame.Generally, foreign DNA is inserted at one or more restriction sites ofthe vector DNA, and then is carried by the vector into a host cell alongwith the transmissible vector DNA. A segment or sequence of DNA havinginserted or added DNA, such as an expression vector, can also be calleda “DNA construct.” A common type of vector is a “plasmid”, whichgenerally is a self-contained molecule of double-stranded DNA, usuallyof bacterial origin, that can readily accept additional (foreign) DNAand which can readily introduced into a suitable host cell. A largenumber of vectors, including plasmid and fungal vectors, have beendescribed for replication and/or expression in a variety of eukaryoticand prokaryotic hosts. The term “host cell” means any cell of anyorganism that is selected, modified, transformed, grown or used ormanipulated in any way for the production of a substance by the cell.For example, a host cell may be one that is manipulated to express aparticular gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays that aredescribed infra. Host cells may be cultured in vitro or one or morecells in a non-human animal (e.g., a transgenic animal or a transientlytransfected animal).

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirusvectors, Drosophila cells (Schneider cells) and expression systems, andmammalian host cells and vectors.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, a gene encoding a protein of interest, e.g., an FGF4 gene, isheterologous to the vector DNA in which it is inserted for cloning orexpression, and it is heterologous to a host cell containing such avector, in which it is expressed, e.g., a CHO cell. with a differentgene that the one it is operatively associated with in nature.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g., DNA, or any process, mechanism or result of such achange. This includes gene mutations, in which the structure (e.g., DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., RNA, protein orenzyme) expressed by a modified gene or DNA sequence. The term “variant”may also be used to indicate a modified or altered gene, DNA sequence,RNA, enzyme, cell, etc.; i.e., any kind of mutant. For example, thepresent invention relates to altered or “chimeric” RNA molecules thatcomprise an rRNA sequence that is altered by inserting a heterologousRNA sequence that is not naturally part of that sequence or is notnaturally located at the position of that rRNA sequence. Such chimericRNA sequences, as well as DNA and genes that encode them, are alsoreferred to herein as “mutant” sequences.

“Sequence-conservative variants” of a polynucleotide sequence are thosein which a change of one or more nucleotides in a given codon positionresults in no alteration in the amino acid encoded at that position.

“Function-conservative variants” of a polypeptide or polynucleotide arethose in which a given amino acid residue in the polypeptide, or theamino acid residue encoded by a codon of the polynucleotide, has beenchanged or altered without altering the overall conformation andfunction of the polypeptide. For example, function-conservative variantsmay include, but are not limited to, replacement of an amino acid withone having similar properties (for example, polarity, hydrogen bondingpotential, acidic, basic, hydrophobic, aromatic and the like). Aminoacid residues with similar properties are well known in the art. Forexample, the amino acid residues arginine, histidine and lysine arehydrophilic, basic amino acid residues and may therefore beinterchangeable. Similar, the amino acid residue isoleucine, which is ahydrophobic amino acid residue, may be replaced with leucine, methionineor valine. Such changes are expected to have little or no effect on theapparent molecular weight or isoelectric point of the polypeptide. Aminoacid residues other than those indicated as conserved may also differ ina protein or enzyme so that the percent protein or amino acid sequencesimilarity (e.g., percent identity or homology) between any two proteinsof similar function may vary and may be, for example, from 70% to 99% asdetermined according to an alignment scheme such as the Cluster Method,wherein similarity is based on the MEGALIGN algorithm.“Function-conservative variants” of a given polypeptide also includepolypeptides that have at least 60% amino acid sequence identity to thegiven polypeptide as determined, e.g., by the BLAST or FASTA algorithms.Preferably, function-conservative variants of a given polypeptide haveat least 75%, more preferably at least 85% and still more preferably atleast 90% amino acid sequence identity to the given polypeptide and,preferably, also have the same or substantially similar properties(e.g., of molecular weight and/or isoelectric point) or functions (e.g.,biological functions or activities) as the native or parent polypeptideto which it is compared.

Thus, for example, in particular embodiments wherein the polypeptidesare FGFR polypeptides, function-conservative variants may not only havebetween at least 75% and at least 90% amino acid sequence identity to agiven FGFR, but preferably also have similar properties, such asconserved domains (e.g., as in a D1, D2 or D3 domain, described supra)and/or similar biological function or activities, such as a tyrosinekinase activity and/or the ability to stimulate activities associatedwith FGF signaling (e.g., mitogenesis or angiogenesis).

Similarly, in embodiments wherein a polypeptide is an FGF ligand,function-conservative variants may not only have between at least 75%and at least 90% amino acid sequence identity to a given FGF, butpreferably also have similar properties. For example, afunction-conservative variant of an FGF ligand preferably binds to thesame FGF receptor as the FGF ligand. (preferably, but not necessarilywith the same or a similar affinity; e.g., preferably with at least 50%of the binding affinity, more preferably with at least 70% of thebinding affinity, and still more preferably with at least 80% or atleast 90% of the binding affinity). Preferably, by binding to the FGFRpolypeptide, a function-conservative variant will also stimulate a samebiological function or activity that is associated with binding of theFGF ligand to the receptor, including any of the functions or activitiesdescribed, supra, for an FGF receptor.

The term “homologous”, in all its grammatical forms and spellingvariations, refers to the relationship between two proteins that possessa “common evolutionary origin”, including proteins from superfamilies(e.g., the immunoglobulin superfamily) in the same species of organism,as well as homologous proteins from different species of organism (forexample, myosin light chain polypeptide, etc.; see, Reeck et al., Cell1987, 50:667). Homologous proteins of the invention therefore includevarious FGF proteins and polypeptides derived from the same species oforganism (i.e., the FGF family of polypeptides, including FGF1-FGF22),and also FGF proteins and polypeptides derived from different species oforganisms. Similarly, homologous proteins of the invention also includevarious FGFR proteins and polypeptides derived from the same species(i.e., the FGFR family, including FGFR1-4) or from different species oforganisms.

Such proteins (and their encoding nucleic acids) have sequence homology,as reflected by their sequence similarity, whether in terms of percentidentity or by the presence of specific residues or motifs and conservedpositions. For instance, referring agin to particular embodiments wherehomologous polypeptides are FGF and/or FGFR polypeptides, homologouspolypeptides in either the same or in closely related species oforganisms (for example, between mammals such as mice and humans)typically share greater than 50% sequence identity, more preferablyshare at least about 60 to 65% sequence identity, and still morepreferably share at least about 75% to 80% sequence identity. Homologouspolypeptides between closely related species of organisms may also becross reactive in both species of organisms. For example, an FGF fromone species of organism may bind to and/or activate an FGF receptorpolypeptide from a different species of organism and, moreover, an FGFreceptor from a first species of organism may stimulate a activityassociated with FGF signalling (e.g., mitogenesis or angiogenesis) in acell from a different species of organism (for example, when theheterologous FGFR polypeptide is recombinantly expressed in that cell).

The term “sequence similarity”, in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin(see, Reeck et al., Cell 1987, 50:667). However, in common usage and inthe instant application, the term “homologous”, particularly whenmodified with an adverb such as “highly”, may refer to sequencesimilarity and may or may not relate to a common evolutionary origin.

In specific embodiments, two nucleic acid sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and morepreferably at least about 90% or at least about 95% of the nucleotidesmatch over a defined length of the nucleic acid sequences, as determinedby a sequence comparison algorithm known such as BLAST, FASTA, DNAStrider, CLUSTAL, etc. An example of such a sequence is an allelic orspecies variant of the specific genes of the present invention.Sequences that are substantially homologous may also be identified byhybridization, e.g., in a Southern hybridization experiment under, e.g.,stringent conditions as defined for that particular system.

Similarly, in particular embodiments of the invention, two amino acidsequences are “substantially homologous” or “substantially similar” whengreater than 80% of the amino acid residues are identical, or whengreater than about 90% of the amino acid residues are similar (i.e., arefunctionally identical). Preferably the similar or homologouspolypeptide sequences are identified by alignment using, for example,the GCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison Wis.) pileup program, or using any of the programsand algorithms described above (e.g., BLAST, FASTA, CLUSTAL, etc.).

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 10, preferably at least 15, and more preferably atleast 20 nucleotides, preferably no more than 100 nucleotides, that ishybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNAmolecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin or a fluorescent dye (forexample, Cy3 or Cy5) has been covalently conjugated. In one embodiment,a labeled oligonucleotide can be used as a probe to detect the presenceof a nucleic acid. In another embodiment, oligonucleotides (one or bothof which may be labeled) can be used as PCR primers; e.g. for cloningfull length or a fragment of either an FGF or an FGFR nucleic acid, orto detect the presence of nucleic acids encoding either an FGF or anFGFR polypeptide. In a further embodiment, an oligonucleotide of theinvention can form a triple helix with a an FGF or an FGFR DNA or RNAmolecule. Generally, oligonucleotides are prepared synthetically,preferably on a nucleic acid synthesizer. Accordingly, oligonucleotidescan be prepared with non-naturally occurring phosphoester analog bonds,such as thioester bonds, etc.

5.3 X-ray Crystallography Definitions

The present invention also uses techniques of conventional X-raycrystallography. These techniques are well known and are within theroutine skill of the art. Such techniques are described more fully inthe literature. See, for example, Cantor & Schimmel, BiophysicalChemistry 1980 (Vols. I-III) W. H. Freeman and Company (particularlyChapters 1-13 in Vol. I, and Chapter 13 in Vol. II).

The term “crystal” refers, generally, to any ordered (or at leastpartially ordered) three-dimensional array of molecules. Preferably, theordering of molecules within a crystal is at least sufficient to producea sharp X-ray diffraction pattern so that the molecules'three-dimensional structure may be determined.

The molecules in a crystal may be of any type, and it will be understoodthat a crystal may contain molecules of only one type or may comprise aplurality of different types of molecules. In preferred embodiments,crystals of the present invention comprise at least one biomolecule,such as a protein, or a fragment thereof. Crystals of the invention mayeven comprise a complex or assembly of two or more proteins or otherbiomolecules. For example, a crystal may comprise two differentproteins, such as a receptor and a ligand, or a crystal may comprise twomore molecules of the same protein bound together, e.g., to form a dimeror other multimer complex. Typically, crystals that contain biologicalmolecules such as proteins will contain other molecules as well, suchmolecules of solvent (e.g., water molecules) and/or salt. Othermolecules such as drugs, drug candidates or compounds that bind to theprotein may also be present in a crystal.

It will be understood by a skilled artisan that crystals of theinvention comprises a “unit cell”, or basic parallelepiped shaped blockdefined by vectors denoted a, b and c. The entire volume of a crystalmay be constructed by the regular assembly of such blocks or “lattices”.A crystal is also defined by the overall symmetry of elements (i.e.,molecules) within the cell, which is referred to as the “space group.”Thus, a crystal's space group is defined by symmetry relations withinthe molecules making up the unit cell. The “asymmetric unit” is thesmallest possible unit from which the crystal structure may be generatedby making use of the symmetric relations defining the space group.

The term “structure coordinates” or “structure” refers to mathematicalcoordinates that define the position of atoms in a molecule or in anassembly of molecules in three-dimensional space (for example, withinthe asymmetric unit of a crystal). Structure coordinates may be computedor otherwise determined using any information related to the threedimensional arrangement of atoms in a molecule. However, in preferredembodiments of the invention a structure is derived from equations thatare related to patterns obtained on diffraction of a monochromatic beamof X-rays by the atoms (which, in such embodiments, may also be referredto as “scattering centers”) in a crystal. Typically, such diffractiondata is used to calculate an “electron density” map of the crystal'sasymmetric unit, and these maps are used, in turn, to establishpositions of the individual atoms.

“Heavy atom derivatization” refers to a method of producing chemicallymodified forms of a crystal (typically a crystal of a protein or otherbiopolymer), in which the crystal may be soaked in a solution containingheavy metal atom salts or organometallic compounds that can diffusethrough the crystal and bind to the surface of the protein orbiopolymer. The location(s) of one or more heavy meatl atoms in thecrystal may then be determined by X-ray diffraction analysis of thesoaked crystal, and this information may be used to facilitateconstruction of the three-dimension structure of the protein or othermolecules contained in the crystal.

“Molecular replacement” refers to a method wherein a preliminarystructure coordinates are generated for molecules in a crystal whosestructure coordinates are not known. Generally, molecular replacementinvolves orienting and/or positioning another, preferably similarmolecule (such as a homologous protein) whose structure coordinates areknown. Phases for an X-ray diffraction pattern may then be determinedfor the preliminary structure, and these phases can then be combinedwith actual X-ray diffraction intensities that are observed for thecrystal whose structure coordinates are not known, to determine itsstructure.

5.4 Detailed Description of Invention

The present invention relates to a particular FGF ligand, known in theart as FGF4 and described in the Examples, infra. Such FGF polypeptidesgenerally comprise the amino acid sequence set forth in FIG. 2, or asimilar (i.e., homologous or highly-homologous) amino acid sequence. Inother preferred embodiments, the methods and compositions of theinvention relate to certain variants of the FGF4 polypeptide,hereinafter alternatively referred to as FGF4 mutants or mutantpolypeptides. Preferred variants of the FGF4 polypeptide are ones havingaltered binding affinities either for heparin, for an FGF receptor(e.g., FGFR1) or for both FGFR and heparin. For instance, the Examples,infra, describe several specific variants of FGF4 having one or moreamino acid residue substitutions, insertions or deletions that alter thepolypeptides' binding affinities for heparin and/or for an FGFR (inparticular, for FGFR1). These variant FGF4 polypeptides are thereforeconsidered part of the present invention.

The Examples also provide a description of model three-dimensionalstructures that describe the binding of FGF4 to an FGF receptor and toheparin. Using these models, a skilled artisan may readily identifyamino acid residues of either FGF or FGFR that may be modified toproduce molecules with greater or lower binding affinities thatwild-type FGF4 and FGFR. These molecules are useful agonist andantagonist for FGF ligand-receptor binding and/or for bioactivitiesassociated therewith. Accordingly, the compositions and methods of theinvention include screening methods that identify such molecules,preferably using the models of the present invention and/or by modifyingkey amino acid residues identified in the Examples, below.

The present invention identifies three regions or domains, hereinafterreferred to as (1) the primary binding site, which is the surfacethrough which one FGF4 molecule binds to one FGFR molecule and forms acomplex, (2) the secondary binding site, which is the surface whichbinds to a second FGFR molecule in the context of an FGF4: FGFR-heparindimer and (3) the heparin binding site, which binds heparin. The primarybinding site for the FGF4 polypeptide involves amino acid residuenumbers (from FIG. 2) 80-82, 84, 87, 92, 95, 108, 117, 119-124, 126,129, 136, 151, 159, 161, 165-167 and 203-205. The heparin binding siteinvolves amino acid residue numbers 89, 90, 182-184, 186, 189, 192 and197-199. The secondary binding site involves amino acid residue numbers162, 163, 195, 196 and 201. These regions are shown in FIG. 6. Mutationin at least one of the amino acids in these regions may lead to theproduction of either antagonists or agonists. Such polypeptides can betested for their ability to stimulate DNA synthesis, in the presence orin the absence of heparin, and/or for their ability to bind to receptorson CHO cells, as described below. Non-limiting examples of mutant FGF4polypeptides produced pursuant to the present invention are shown inTable 2.

The identification of the three major domains or binding sites of FGF4that are important for receptor binding and receptor activation couldlead to the rational design of mutated FGF4 forms with alteredbiological activity or function. Such mutant forms of FGF4 couldpossess 1) higher receptor binding affinity and therefore higherbiological potency, 2) the ability to bind to the receptor withoutinducing receptor dimerization, therefore acting as FGF receptorantagonists, or 3) the ability to interact with different forms ofheparin or heparin sulfate proteoglycans, therefore possibly modifyingits spectrum of action in a biological context.

This could be accomplished by mutating selected amino acids in theprimary binding site, and the secondary binding site, respectively toamino acids which would make stronger bonds with the correspondingreceptor region and thus create new FGF4s with higher biologicalactivity. An example of such mutations culd be changing F129 to Y or I,R205 to M or L in the primary binding site domain A, or H201 to L in thesecondary binding site. On the other hand, by mutating amino acids inthe secondary binding site (the dimerization domain) to Alanine or otheramino acids it should be possible to create mutants FGF4 poly-peptideswhich bind the receptor as a monomer, but are incapable of promotingreceptor dimerization, and thus behave as FGF4 receptor antagonists.Furthermore, mutations of the amino acids making up the heparin bindingdomain would result in lower or higher heparin binding activity.Mutations in this domain could be combined with mutations in the primarybinding site to produce a more active FGF4, or they could be combinedwith mutations in the secondary binding site to produce a betterantagonist, since it is known that heparin (that binds to both receptorand FGF ligand) contributes to the formation of stable, active FGF4/FGFRdimers.

Alternatively, one could search databases of chemical or peptidelibraries well known to those of ordinary skill in the art, to identifysmall molecules (peptides) which could bind to the primary or secondarybinding sites of FGF4, and thus would inhibit binding of the FGF4 ligandto its receptors.

A further application of the findings presented herein is the screeningof the above mentioned databases for molecules which mimic the structureof the primary or secondary binding sites of FGF4. Such molecules wouldbe expected to bind to FGF4 receptors, but would be unable to activatesignaling through the receptor. They would however compete with thebinding of FGF4 to its receptor, or with its ability to induce receptordimerization, and thus would act as FGFR antagonists.

FGF4 mutant polypeptides for use in the present invention can beproduced as described below, e.g., by site-directed mutagenesis of thenucleic acid encoding FGF4. subdloned in an expression vector andpurified as described below. Antagonists can be used to treat patientssuffering from FGF4-mediated illnesses such as bladder cancer. Agonistscan be employed when using FGF4 for its angiogenic or wound healingproperties. The nucleic acid encoding FGF4 can be obtained as describedin U.S. Pat. Nos. 5,750,659 and 5,459,250. Either full length FGF4 ortruncated FGF4 may be used.

6. EXAMPLES

The present invention is also described by means of particular examples.However, the use of such examples anywhere in the specification isillustrative only and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to any particular preferred embodiments described herein.Indeed, many modifications and variations of the invention will beapparent to those skilled in the art upon reading this specification andcan be made without departing from its spirit and scope. The inventionis therefore to be limited only by the terms of the appended claimsalong with the fill scope of equivalents to which the claims areentitled.

To explore the structural determinants of FGF4 involved in receptor andheparin binding, the crystal structure of FGF4 was determined, asdescribed here, at 1.8 Å resolution. As expected, FGF4 adopts aβ-trefoil fold similar to other FGFs. Superimposition of FGF4 structureonto FGF2 bound to FGFR1 and heparin allows the identification ofreceptor and heparin binding sites in FGF4. Mutation of several key FGF4residues, observed to interact with FGFR1 in the model described here,produces variant FGF4 ligands that have reduced receptor bindingaffinities and extremely low mitogenic potential. Significantly, theobserved interactions between FGF4 and FGFR1 D3 provide a potentialbasis for preferential affinity of FGF4 towards IIIc splice variants ofFGFR1-3. Moreover, the presented modeling studies along with mutationaldata suggest a two step model for FGF-FGFR binding that involves initialformation of a crucial FGF-D2 interface stabilized by heparin bindingfollowed by secondary FGF-D3 interactions.

6.1 Materials and Methods

Protein expression and purification of FGF4. DNA fragments generated bypolymerase chain reaction (PCR) of human FGF4 cDNA (encoding residuesGly79 to Leu206) were subcloned into the pET-15b bacterial expressionvector using NcoI and XhoI cloning sites. The resulting construct(FGF4-pET15b) was transformed into the BL21 (DE3) bacteria and FGF4expression was induced with 1 mM Isopropyl-1-thio-β-D-galactopyranosidefor 5 hours. The bacteria were then centrifuged and subsequently lysedin a 25 mM Na/K phosphate buffer (pH 7.5) containing 300 mM NaCl using aFrench Cell Press. The N-terminal truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) wasfound primarily in the insoluble fraction and was extracted in 25 mMNa/K phosphate buffer (pH 7.5) containing 1 M NaCl at 4° C. overnight.Following centrifugation, soluble FGF4 was diluted 5 times with 25 mMNa/K phosphate buffer (pH 7.5) and loaded onto a Source S column(Pharmacia). Bound FGF4 was eluted by a linear gradient of NaCl to 1 Min a 25 mM Na/K phosphate buffer (pH 7.5). Matrix-assisted laserdesorption ionization mass spectrometry of the purified FGF4 gave amolecular mass of 14,244 daltons (Da) (calculated mass 14,409 Da). Themass difference was due to the cleavage of the initiation methionine anda point mutation (Ser182Gly) resulting from PCR. This mutation had noeffect on FGF4 biological activity.

Crystallization and data collection. Crystals of FGF4 were grown byvapor diffusion at 20° C. using the hanging drop method. Briefly, 2 μlof protein solution (2 mg/ml in 25 mM HEPES-NaOH buffer (pH 7.5)containing 150 mM NaCl) were mixed with an equal volume of thecrystallization buffer (30% Polyethylene glycol 8000, 0.2 M ammoniumsulfate). FGF4 crystals thus obtained belong to the orthorhombic spacegroup P2₁2₁2₁ with unit cell dimensions a=40.37 Å, b=53.3 Å, and c=56.23Å. There is one molecule of FGF4 in the asymmetric unit with a solventcontent of approximately 43%. Diffraction data were collected from aflash-frozen (in a dry nitrogen stream using mother liquor containing10% glycerol as cryo-protectant) crystal on an R-Axis IV image platedetector at Beamline X4-A at the National Synchrotron Light Source,Brookhaven National Laboratory. Data were processed using DENZO andSCALEPACK (see, Otwinowski & Minor, Methods Enzymol. 1997, 276:307-326).

Structure determination and refinement. A molecular replacement solutionwas found for one copy of FGF4 in the asymmetric unit using the programAmoRe (Navaza, Acta Crystallogr. Sect. A 1994, 50:157-163) and thestructure of FGF2 (Protein Data Bank entry 2FGF; see also, Zhang et al.,Proc. Natl. Acad. Sci. U.S.A. 1991, 88:3446-3450) as the search model.Simulated annealing and positional/B-factor refinement were performedusing CNS (Bruenger et al, Acta Crystallogr. Sect. D 1998, 54:905-921).Bulk solvent and anisotropic B-factor corrections were applied. Modelbuilding into 2F₀-F_(c) and F_(o)-F_(c) electron density maps wasperformed with the program O (Jones et al., Acta Crystallogr. Sect. A1991, 47:110-119). The atomic model contains residues 79 to 206 of FGF4,3 sulfate ions, and 96 water molecules. The average B-factor is 10.5 Å²for the FGF4 molecule, 40.5 Å² for the sulfate ions, and 17.5 Å² for thewater molecules.

Production of mutant FGF4 proteins. Alanine substitutions wereintroduced into the N-terminal truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) by PCRsite-directed mutagenesis (Quik Change™, Stratagene) with theFGF4-pET15b expression plasmid (see above for description) as thetemplate and the following mutant oligos as primers:

Y87A: 5′-AAGCGGCTGCGGCGGCTCGCATGCAACGTGGGCATCGGC-3′ F129A:5′-GCGTGGTGAGCATCGCCGGCGTGGCCAGCCGG-3′ F151A5′-CTATGGCTCGCCCTTCGCGACCGATGAGTGCACGTTC-3′ E159A:5′-GATGAGTGCACGTTCAAGGCCATTCTCCTTCCCAAC-3′ Y166A:5′-CTCCTTCCCAACAACGCGAACGCGTACGAGTCC-3′ L203:5′-CCATGAAGGTCACCCACTTCGCCCCTAGGCTGTGACCC-3′ R205A:5′-CCCACTTCCTCCCCGCGCTGTGACCTTCCAGAGG-3′ N89A:5′-CGGCGGCTCTACTGCGCCGTGGGCATCGGCTTC-3′ K198A:5′-GTGTCGCCCACCATGGCGGTCACCCACTTCCTC-3′ K183A/K15′-GCCCTGAGCGCGAATGGGAAGACCGCGAAGGGGAAC-3′ 88A: R103A:5′-GCGCTCCCCGACGGCGCCATCGGCGGCGCGCAC-3′ K144A:5′-ATGAGCAGCAAGGGCGCGCTCTATGGCTCGCCC-3′ N89A/K19 Alanine substitutionwere introduced into the N- 8A/K1893 terminally trunacted FGF4N89A/K198A mutant as a A/K188A: template by PCR site-directedmutagenesis with sense and antisense oligonucleotides containingK183A/K188A mutations: 5′-GCCCTGAGCGCGAATGGGAAGACCGCGAAGGGGAAC-3′ E117A5′-CGCGACAGCCTGCTGGCGCTCTCGCCCGTGGAG-3′ L162A5′-TTCAAGGAGATTCTCGCTCCCAACAACTACAA-3′ H201A5′-ACCATGAAGGTCACCGCCTTCCTCCCAGGCT-3′ P163A5′-GGAGATTCTCCTTGCCAACAACTACAACGCC-3′ P195A5′-GGGAACCGAGTGTCGGCCACCATGAAGGTCACC-3′ T196A5′-GGGAACCGAGTGTCGCCGCCATGAAGGTCACCCACTTCC-3′The presence of the mutations was confirmed by sequencing. MutantFGF4-pET15b plasmids were transformed into E. coli strain BL21 (DE3).Expression of the FGF4 proteins was induced as described above.Following centrifugation, cells expressing wild type and various mutantFGF4 proteins were suspended in a 50 mM HEPES-NaOH buffer (pH 7.4)containing 1 M NaCl and protease inhibitors (100 μg/ml PMSF, and 2 μg/mlAprotinin) and disrupted by sonication. Lysates were left at 4° C.overnight in order to salt-extract the FGF4 proteins from particulatefractions. Following centrifugation supernatants containing soluble FGF4proteins were diluted 4 times with 50 mM HEPES-NaOH buffer (pH 7.4), andloaded onto heparin-Sepharose columns. After washing the columns with 50mM HEPES-NaOH (pH 7.4) buffer containing 250 mM NaCl, the FGF4 proteinswere eluted by a 50 mM HEPES buffer (pH 7.4) containing 1.5 M NaCl.Fractions were analyzed by SDS-PAGE (15%) and the purity of the FGF4proteins was accessed by staining with Coomassie Blue R-250.

DNA synthesis assay. NIH3T3 cells were seeded at a density of 3×10⁴cells/well in 24-wells plates in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% calf serum. The following day the mediumwas replaced with DMEM containing only 0.5% calf serum and the cellswere allowed to reach quiescence for 48 hours. Thereafter, serialdilutions of wild-type full length FGF4 (Ala³¹-Leu²⁰⁶), N-terminaltruncated wild type or mutant FGF4 (Gly⁷⁹-Leu²⁰⁶) were added for 18hours. Cells were then labeled with 1 μCi of ³H-thymidine for 6 hours,washed with Tris-HCl buffered saline (pH 7.5), and lysed with 0.5 MNaOH. The lysates were then neutralized with 0.5 HCl and theradioactivity incorporated into the acid-precipitable material wasmeasured using a β-counter (LKB, Pharmacia). Each assay was performed intriplicate.

Receptor bintdinig assay. N-terminal truncated FGF4 (Gly⁷⁹-Leu²⁰⁶) wasradio-iodinated by the chloramine T method using a previously describedprotocol (Bellosta et al., J. Cell Biol. 1993, 121:705-713). The labeledFGF4 were separated from free iodine over a Sephadex G-25 column, whichwas previously equiliberated in phosphate buffered saline (PBS)containing 1% bovine serum albumin. CHO cells, over-expressing FGFR2(Mansukhani et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89:3305-3309),were seeded at 1×10⁶ cells/well in 6 well plates in DMEM containing 10%fetal calf serum. The following day the medium was removed and the cellswere allowed to bind labeled FGF4 (specific activity 2.5×10⁴ cpm/ng) inDMEM containing 25 mM HEPES-NaOH (pH 7.4), 15% gelatin, 10 μg/mlheparin, and increasing concentrations of the wild type or mutant FGF4proteins for 2 hours at 4° C. Cells were then washed several times withice-cold Tris-HCl buffered saline (pH 7.5) and the receptor-boundradio-labeled FGF4 was released using a 50 mM Sodium Acetate buffer (pH4.0) containing 2 M NaCl. Radioactivity was measured using a γ-counter(LKB-Pharmacia). Binding assays were done in duplicate.

6.2 Results and Discussion

Structure determination. The mature, secreted form of human FGF4 spansamino acids Ala31 to Leu206 (Bellosta et al., J. Cell Biol. 1993,121:705-713). Based on the crystal structure of FGF2 (Eriksson et al.,Proc. Natl. Acad. Sci. U.S.A. 1991, 88:3441-3445; Zhang et al., Proc.Natl. Acad. Sci. U.S.A. 1991, 88:3446-3450; Zhu et al., Science 1991,251:90-93), the β-trefoil core of FGF4 is expected to start at Leu83(Pro29 in FGF2). Recent crystal structures of three different FGF-FGFRcomplexes have revealed that the residues immediately preceeding theβ-trefoil core in FGF1 and FGF2 are involved in FGFR binding (Plotnikovet al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000, 101:413-424;Springer et al., J. Biol. Chem. 1994, 269:26879-26884). These residuescorrespond to amino acids Gly79 to Arg82 of FGF4 (FIG. 2). Thus, tomaximize the likelihood of obtaining diffracting crystals withoutjeopardizing the biological activity of FGF4, an N-terminal truncatedFGF4 polypeptide containing residues Gly79 to Leu206 (Gly⁷⁹-Leu²⁰⁶) wascrystallized. Truncated FGF4 was expressed in E. coli and purified tohomogeneity (see, Subsection 6.1, supra). The mitogenic activity of thetruncated FGF4 on NIH3T3 cells was only slightly lower than that of themature FGF4, indicating that the truncated FGF4 contains the majority ofreceptor binding sites. Crystallization trials with FGF4 producedorthorhombic crystals with one molecule per asymmetric unit. The crystalstructure of FGF4 was solved by molecular replacement as described,supra, and refined to 1.8 Å resolution with an R-value of 19.4% (freeR-value of 20.7%). The atomic model for FGF4 consists of one FGF4molecule (residues 79 to 206), 3 sulfate ions and 96 water molecules.The coordinates for the FGF4 crystal structure are given in theaccompanying Appendix B, infra, in PDB file format. Data collection andrefinement statistics are given below in Table 1.

TABLE 1 Summary of crystallographic analysis I. Data CollectionStatistics: Resolution Reflections Completeness R_(sym) ^(a) Signal (Å)(total/unique) (%) (%) (<|/σ|>) 25.0-1.8 40562/11590 99.6 (995)^(b) 5.1(10.9)^(b) 16.5 II. Refinement Statistics:^(c) Root-mean-squareDeviations Resolution R_(cryst)/R_(free) ^(d) Bonds Angles B-factors^(e)(Å) Reflections (%) (Å) (°) (Å²) 25.0-1.8 11488 19.4/20.7 0.005 1.311.00$\;^{a}R_{sym} = {100 \times {\sum\limits_{hkl}{\sum\limits_{i}{{{{l_{i}({hkl})} - \left\langle {l({hkl})} \right\rangle}}/{\sum\limits_{hkl}{\sum\limits_{i}{{l_{i}({hkl})}.}}}}}}}$^(b)Value in parentheses is for the highest resolution shell: 1.86-1.8Å. ^(c)Atomic model: 994 protein atoms, 3 SO₄ ²⁻ ions, and 42 watermolecules.${\;^{d}R_{{cryst}/{free}} = {100 \times {\sum\limits_{hkl}{{{{{F_{o}({hkl})}} - {{F_{c}({hkl})}}}}/{\sum\limits_{hkl}{{F_{o}({hkl})}}}}}}},$where F_(o) (>0σ) and F_(c) are the observed and calculated structurefactors. 5% of the reflections were used for calculations of R^(free).^(e)For bonded protein atoms.

Description of the structure. As expected by sequence similarity(Vainikka et al., EMBO J. 1993, 11:4273-4280), FGF4 adopts a β-trefoilfold conformation (FIG. 1). Superimposition of the Cα traces locatedwithin the β-trefoil core of FGF4 with those of FGF1 (Zhu et al.,Science 1991, 251:90-93) and FGF2 (Zhang et al., Proc. Natl. Acad. Sci.U.S.A. 1991, 88:3446-3450) gives a root-mean-square (rms) deviation ofonly 0.86 Å (122 common Cα atoms) and 0.76 Å (123 common Cα atoms),respectively. Within the β-trefoil core, the major differences betweenthe FGF4 structure and the structures of FGF1 and FGF2 are theconformations of the β1-β2, β3-β4 and β9-β10 loops. These loops vary inboth length and sequence among the various members of the FGF family(FIG. 2). In FGF4, the β1-β2 loop is one residue longer than thecorresponding loops of FGF1 and FGF2 (FIG. 2). In contrast, the β3-β4loop in FGF4 is shorter by one residue than the corresponding loops inFGF1 and FGF2. Like FGF2, the β9-β10 loop in FGF4 is shorter by tworesidues than in FGF1 (FIG. 2).

As with the structures of free FGF1 and FGF2 (Zhu et al., Science 1991,251:90-93), a sulfate ion is bound to the predicted high affinityheparin-binding site of FGF4. In addition, two other sulfate ions arecoordinated by FGF4 residues, whose corresponding residues in FGF1 andFGF2 have not been observed to bind sulfate ions (see, FIG. 6).

Receptor binding sites and specificity. To identify potential receptorbinding sites in FGF4, an FGF4-FGFR1 model was constructed bysuperimposing the FGF4 structure onto the FGF2 structure bound to theligand binding portion of FGFR1 consisting of Ig-like domains 2 (D2) and3 (D3) (FIG. 3). Careful inspection of the FGF4-FGFR1 interface showedthat the majority of the interactions between FGF4 and FGFR1 in theFGF4-FGFR1 model could be accommodated with minor adjustments of FGF4side chain rotamers. Three loop regions, the β1-β2 and β9-β10 loops(within the β-trefoil core) and the N-terminus (outside the β-trefoilcore), sterically clash with the receptor. In the present crystalstructure these loop regions are involved in crystal lattice contacts,implying that the present conformations of these loops are dictated bythe lattice contacts. However, upon FGFR binding these regions areexpected to undergo changes in backbone conformation to allow anengagement with FGFR1 to occur.

At the FGF4-D2 interface three highly conserved solvent-exposed FGF4residues, Tyr87, Tyr166 and Leu203, are predicted to pack against ahighly conserved hydrophobic surface consisting of Ala167, Pro169, andVal248 at the bottom of D2 in FGFR1 (data not shown). Significantdifferences between FGF4 and FGF2 at the FGF-D2 interface are thesubstitutions of Phe40 and Met151 in FGF2 with His95 and Arg205 in FGF4(FIG. 2). These substitutions may indicate a weaker hydrophobic FGF4-D2interface compared to the FGF2-D2 interface. At the FGF4-linkerinterface, Asn167, also highly conserved among FGFs (FIG. 2), isexpected to make hydrogen bonds with the FGFR-invariant arginine (Arg250in FGFR1) in the D2-D3 linker region.

To provide experimental support for the described interactions betweenFGF4 and FGFR1 at the FGF4-D2 interface, Tyr87, Tyr166, Leu203 andArg205 were individually mutated to alanine in the N-terminal truncatedFGF4 construct (Gly⁷⁹-Leu²⁰⁶). Mutant FGF4 proteins were expressed in E.coli, purified to near homogeneity as described, supra, and were assayedfor the ability to induce DNA synthesis in NIH 3T3 fibroblasts. As shownin FIG. 4 and in Table 2, below, the Y87A, Y166A and L203A mutant FGF4proteins were severely compromised in their ability to induce DNAsynthesis, while the R205A mutant showed a more modest decrease in theinduction of DNA synthesis. Thus, these data confirm the observedinteractions between FGF4 and D2 in the FGF4-FGFR1 model, and indeed thecorresponding 4 residues in FGF2 had been previously shown to be alsoimportant for biological activity (Springer et al., J. Biol. Chem. 1994,269:26879-26884).

TABLE 2 Summary of DNA Synthesis Activity and Receptor Binding Affinityof Mutant FGF4 Polypeptides DNA Synthesis NIH 3T3 cells DNA synthesisassay Receptor Binding (ED₅₀ mutant/ 32D-FGFR2 cells CHO-FGFR2 cellsProtein ED₅₀ wild-type)^(a) ED50mut/ED50wt (IC₅₀ mutant/IC₅₀wild-type)^(b) (Gly⁷⁹-Leu²⁰⁶) −heparin +heparin −heparin +heparin+heparin wild-type 1 1 0 1 1 Y87A >500 500 >100 F129A >500 >500 >100F151A 1000 4 0 200 2.5 E159A >1000 80 0 >1000 5.0 Y166A 500 >500 >100L203A >500 >500 >100 R205A 25 2.5 2.5 N89A/K198A 10 10 0 65 NDK183A/K188A 70 13 0 125 ND R103A/K144A 1 1 ND N89A/K198A/K >1333 2750 >1000 >1000 183A/K188A E117A ≈1 ≈1 0 7 7 L162A 600 158 0 >1000 >1000H201A >666 125 >1000 P163A 10 4.6 P195A 8 4.6 T196A 5.5 4 ^(a)ED₅₀(Effective Dose) is the dose of FGF4 necessary to reach 50% of maximumDNA synthesis obtained with the wild type FGF4. ^(b)IC₅₀ (InhibitoryConcentration) is the concentration of FGF4 required to compete 50% ofbinding of labeled wild type FGF4 to FGFR2. ^(c)ND (Not Determined).^(d)32D is a murine hematopoietic cell line, which does not express FGFreceptors or heparin sulfate proteoglycans. As a consequence, even whentransfected to express FGF receptors (as in the example, 32D-FGFR2),they require exogenous heparin to be stimulated to proliferate by FGF(Mansukhani et al, PNAS 89: 3305, 1992). In the absence of heparin, FGFsare not mitogenic in these cells (expressed as 0 activity).

Interactions between FGF4 and D3 occur at the upper part of D3 andinvolve mainly the βB′-βC, βC′-βE and βF-βG loops in D3. At theinterface between FGF4 and the βB′-βC loop of D3, Glu159 of FGF4 (anFGF-invariant residue) (FIG. 2) is expected to make a hydrogen bond withGln284 (an FGFR-invariant residue). This prediction is supported by a1,000-fold reduction in the ability of the E159A mutant FGF4 to induceDNA synthesis in living cells (FIG. 5A and Table 2, above).

In contrast to the interface between FGF4 and the βB′-βC loop,interactions between FGF4 and the βC′-βE and βF-βG loops are variable.Significantly, FGF4, like FGF1, has a serine (Ser119) at the positionhomologous to Gln65 of FGF2 (FIG. 2). It has previously been shown thatGln65 of FGF2 makes two hydrogen bonds with Asp320/Asp321 in the βC′-βEloop of FGFR1/FGFR2, and as a result, the βC′-βE loop is ordered in bothFGF2-FGFR1 and FGF2-FGFR2 structures (Plotnikov et al., Cell 1999,98:641-650; Plotnikov et al., Cell 2000, 101;413-424). In contrast, inthe FGF1-FGFR1 structure the βC′-βE loop is disordered because Ser62 ofFGF1 cannot interact with Asp320 of FGFR1 in the βC′-βE loop (Plotnikovet al., Cell 2000, 101:413-424). Thus, by analogy a skilled artisanwould expect that Ser119 of FGF4 will also not make hydrogen bonds withAsp320 located in the βC′-βE loop. However, based on the present model,the side chain of Glu117 in FGF4 could make hydrogen bonds with Lys321located in the βC′-βE loop of FGFR1. This interaction may potentiallycompensate for the inability of FGF4 to engage Asp320 and lead to anordered βC′-βE loop. Moreover, in the FGF4-FGFR1 model, severalsolvent-exposed hydrophobic residues in FGF4 (Val121, Phe129, Phe136,Phe151) are in the vicinity of the βC′-βE loop of FGFR1 and could engagein hydrophobic contacts with Val-316 in the βC′-βE loop of FGFR1. Thesehydrophobic interactions would further contribute to the ordering of theβC′-βE loop in an FGF4-FGFR1 structure. DNA synthesis assays performedusing mutant FGF4 molecules support the aforementioned hypothesis.Substitutions of Phe129 and Phe151 with alanine in FGF4 reduced theability of FGF4 to induce thymidine-incorporation in NIH3T3 cells abouta thousand-fold (Table 2, above). Mutation of the residue correspondingto Phe151 in FGF2 was also shown to be important for FGFR binding (Zhuet al., Protein Eng. 1998, 11:937-940).

Thus, the FGF4-FGFR1 model presented shows that FGF4, like FGF2, mayengage the βC′-βE loop of FGFR1. Consequently, sequence variations inthe βC′-βE loop resulting from alternative splicing should affectFGF4-FGFR binding affinity. A sequence comparison of FGFRs at the βC′-βEloop region demonstrates that Lys321 is conserved only in the IIIcisoforms of FGFR1-FGFR3, providing a potential explanation for reducedaffinity of FGF4 towards the IIIb splice variants of FGFR1-FRFR3 andFGFR4.

Binding of the FGF4 mutants to FGFR2. Mutant FGF4 proteins were testedin a receptor binding assay to confirm that the diminished capacity ofthe mutant FGF4 proteins to induce DNA synthesis is a result of thereduced ability of the mutant FGF4 to interact with FGFR. CHO cellsover-expressing FGFR2 (Mansukhani et al., Proc. Natl. Acad. Sci. U.S.A.1992, 89:3305-3309) were allowed to bind radio-labeled N-terminaltruncated FGF4 (Gly⁷⁹-Leu²⁰⁶) in the presence of increasingconcentrations of unlabeled full length (Ala³¹-Leu²⁰⁶), N-terminaltruncated wild-type or various N-terminal truncated mutant FGF4 proteins(FIG. 4). The N-terminal truncated wild-type FGF4 bound FGFR2 with onlya slightly lower affinity than full length FGF4 (Ala³¹-Leu²⁰⁶),indicating that the majority of receptor binding sites is containedwithin the Gly⁷⁹-Leu²⁰⁶ construct (FIG. 4) Substitutions of Tyr87,Tyr166 and Leu203 with alanine severely reduced the affinity of FGF4towards FGFR2 (FIG. 4 and Table 2, supra), emphasizing the importance ofthe hydrophobic FGF4-D2 interface in providing FGF4-FGFR affinity. Incontrast, the R205A mutant FGF4 showed only a slight reduction in FGFR2binding affinity. The relative decrease in binding affinity of thesemutants towards FGFR2 is consistent with the results of the DNAsynthesis assay, thus implying that the impaired ability of thesemutants to induce DNA synthesis is a consequence of loss of bindingaffinity to FGFR2.

Alanine substitutions of FGF4 residues predicted to interact with D3also reduced the binding affinity of FGF4 for FGFR2. The F129A mutantshowed a large decrease (more than 100-fold) in receptor bindingaffinity, which paralleled the severe impairment of this mutant ininduction of DNA synthesis (FIG. 4 and Table 2, supra). In contrast, theF151A and E159A mutants were only slightly affected (2.5- and 5-fold,respectively) in FGFR2 binding (FIG. 4 and Table 2), yet these mutantswere severely compromised in induction of DNA synthesis (see, Table 2,above). This was particularly unexpected for the E159A mutant as Glu159is highly conserved among FGFs (FIG. 2) and the corresponding glutamicacid in FGF2 (Glu96) was shown to be critical for binding of FGF2 toFGFR1 (Zhu et al., J. Biol. Chem. 1995, 270:21869-21874).

This discrepancy between receptor binding and DNA synthesis data for theF151A and E159A mutants may be due to a difference between theexperimental conditions used for the receptor binding and for DNAsynthesis assays. Since NIH 3T3 cells naturally express cell surfaceHSPG in abundance, they do not require exogenous heparin to respondfully to FGF4. Therefore, exogenous heparin was not included in the DNAsynthesis assays. In contrast, receptor binding assays were alsoperformed in the presence of exogenous heparin to exclude binding of FGFto the abundantly expressed cell surface HSPG. In the absence ofheparin, binding to these low affinity but very abundant receptors cannot easily be distinguished from binding to FGFR.

Since heparin stabilizes FGF-FGFR interactions, it was possible that thepresence of exogenous heparin in the receptor binding assay could havepartially reversed the reduced ability of the F151A and E159A mutants tointeract with FGFR. To test this possibility, the DNA synthesis assayswere repeated in the presence of exogenous soluble heparin. While, asexpected, heparin had no effect on the mitogenic ability of wild typeFGF4, heparin dramatically enhanced the capacity of the F151A and E159Amutants to induce DNA synthesis (FIG. 5A and Table 2, above). Incontrast, addition of heparin had no effect on the Y87A, F129A, Y166Aand L203A mutants and enhanced the ability of R205A to induce DNAsynthesis only by about ten-fold (shown in FIG. 5A, and in Table 2).

Analysis of the location of the various FGF4 mutations in the ternaryFGF4-FGFR1-heparin model provides a potential explanation for thedifferential ability of heparin to rescue only some of the mutants. Boththe F115A and E159A mutations, which display the greatest potentiationupon addition of heparin, affect FGF4 interaction with FGFR D3 (FIG. 2).In contrast, with the exception of the F129A mutant, all the mutationsthat are not rescued by heparin affect FGF4 interaction with FGFR D2.Upon binding of FGF to FGFR, a continuous negatively charged surface isformed by the heparin binding sites of FGF and FGFR D2, to which heparinbinds (Plotnikov et al., Cell 1999, 98:641-650). Simultaneous binding ofthe same heparin polymer to both FGF and FGFR will clearly increaseapparent FGF-FGFR affinity (Schlessinger et al., Mol. Cell 2000,6:743-750). Mutations affecting the FGF-D2 interface will hamper aproductive juxtaposition of the heparin binding sites of FGF and FGFR toform a continuous heparin binding surface, and thus heparin will notreverse the deleterious effects of these mutations. In contrast,mutations affecting the FGF-D3 interface will not interfere with theformation of a productive heparin binding surface by FGF and FGFR D2,and heparin can enhance FGF-FGFR affinity by interacting with both FGFand FGFR D2.

The mutagenesis data suggest that interactions of FGF with FGFR D2provide the primary FGF-FGFR binding affinity. Indeed, Wang et al.(Biochemistry 1999, 38:160-171) have shown that several FGFs can bind tothe isolated D2 domain of FGFR1 in vitro in the presence of heparin.Such teaching, coupled with what is taught, supra, in these examples,indicates that FGFR binds FGF first via D2. Heparin then may stabilizethe FGF-D2 interaction and facilitates formation of a FGF-D3 interface.

Heparin binding sites. Recent biochemical and structural datademonstrate that FGF in the absence of heparin can form an initial lowaffinity complex with FGFR (Pantoliano et al., Biochemistry 1994,33:10229-10248). In the presence of heparin, the low affinity complexesbecome stabilized which then in turn lead to stable 2:2 FGF:FGFRsignaling complexes. The recent crystal structure of a dimeric 2:2:2FGF2:FGFR1:heparin complex provides a molecular basis for how heparinenhances FGF-FGFR affinity and promotes dimerization (Schlessinger etal., Mol. Cell 2000, 6:743-750). Within each ternary 1:1:1FGF:FGFR:heparin complex, heparin makes numerous contacts with theheparin binding residues of FGF and FGFR, thereby increasing theaffinity of FGF towards FGFRs. In addition, heparin also interacts withthe heparin binding residues in D2 of the adjoining FGFR, therebyaugmenting the weak interactions of FGF and FGFR in one ternary complexwith the FGFR in the adjoining ternary complex. Since FGFs differ in theprimary sequences of heparin binding sites, each FGF may requiredifferent heparin motifs (e.g., different sulfation patterns and/orlengths) to exert its optimal biological activities (Faham et al., Curr.Opin. Struct. Biol. 1998, 8:578-586; Schlessinger et al., Mol. Cell2000, 6:743-750).

To evaluate the potential heparin binding sites of FGF4, a dimericFGF4-FGFR-heparin model was generated by superimposing two copies of theFGF4 structure onto the two copies of FGF2 in the dimericFGF2-FGFR1-heparin ternary complex (FIG. 6). FGF4 residues correspondingto the heparin binding residues of FGF2 along with other FGF4 surfaceresidues that could bind heparin were mapped onto the ribbon diagram ofFGF4 (data not shown). With the exception of Lys188 (Lys134 in FGF2) andLys198 (Lys144 in FGF2) the remainder of the heparin binding residuesdiffers between FGF4 and FGF2 (FIG. 2). These differences are likely todetermine the optimal sulfation motifs in heparin that are required tosupport FGF4 or FGF2 biological activities. Interestingly, Asn36 andGln143, two critical heparin binding residues in FGF2, are substitutedby hydrophobic residues Val90 and Met197 in FGF4 (FIG. 2). Therefore,these residues are unable to make hydrogen bonds with the hydroxyl groupand N-sulfate group of ring D of heparin. Moreover, in the model theside chain of Val199 in FGF4 (Ala145 in FGF2) clashes with the N-sulfateof ring D (data not shown). These observations indicate that FGF4 maynot require N-sulfate on ring D for heparin binding. Two othersignificant differences between FGF2 and FGF4 are the substitutions ofLys35 and Lys128 of FGF2 with Asn89 and Ser182, respectively, in FGF4(FIG. 2). Based on the present model, Asn89 and Ser182 would engagebetter the 6-O-sulfate of ring B and the 2-O-sulfate group of ring E(data not shown).

A sulfate ion (provided by the crystallization buffer) is coordinated atthe predicted high affinity heparin-binding site of FGF4 by Lys183 andLys188 (data not shown). These two lysines are expected to bind the2-O-sulfate group of ring E of heparin. In fact the sulfate ion in theFGF4 structure nearly colocalizes with the 2-O-sulfate group of ring Ein the FGF4-heparin model (data not shown). To provide experimentalsupport for the modeled FGF4-heparin interactions, mutant FGF4 proteinswere generated in which FGF4 residues predicted to coordinate the2-O-sulfate of ring E (K183 and K188) or the 6-O-sulfate of ring B (N89and K198) are substituted with alanines. Both the doubly mutatedK183A/K188A and N89A/K198A FGF4 proteins showed diminished ability toinduce DNA synthesis in NIH3T3 cells (see, FIG. 5B and Table 2).Mutations in all four (4) amino acid residues abolishes all FGF4activity (Table 2). Thus, as for FGF2, both the 6-O-sulfate of ring Band the 2-O-sulfate group of ring E of heparin may play important rolesin promoting heparin-dependent FGF-FGFR interaction and dimerization.These data are consistent with findings that a high content in6-O-sulfate groups in heparin is required for specific interaction withFGF4 (Ishihara, Blycolbiology 1994, 4:817-824).

Another sulfate ion is coordinated by the side chains of Arg103, Lys144in the crystal structure of FGF4 (data not shown). Since bound sulfateions in the crystal structures of free FGFs often indicate potentialheparin binding sites in FGFs a doubly mutated R103/K144 FGF4 proteinwas also generated. This mutant FGF4 protein induced DNA synthesis inNIH3T3 cells to a level comparable to wild-type FGF4 (see, Table 2),suggesting that Arg103 and Lys144 most likely do not participate inheparin binding.

Experiments were also performed to determined whether exogenous heparincan compensate for the reduced ability of the K183A/K188A and N89A/K198Amutant FGF4 proteins in the DNA synthesis assay. As shown in FIG. 5B,exogenously added heparin significantly enhanced the ability of theK183A/K188A mutant FGF4 to induce DNA synthesis, but had no effect onthe N89A/K198A mutant. A possible explanation for the differentialeffect of heparin on the activity of these two mutants lies in theheterogenous nature of commercial heparin. It is known that heparin is amixture of oligosaccharides of different length and sulfate contents,generated by the polymerization of repeating disaccharide unitsconsisting of D-glucoseamine (GlcN) and L-iduronic acid (IdoA). Duringbiosynthesis, heparin is sulfated by the sequential actions of threedifferent sulfotransferases, an N-sulfotransferase, a2-O-sulfotransferase and a 6-O-sulfotransferase (Lindahl et al., J.Biol. Chem. 1998, 273:24979-24982). In general, these reactions proceedin the order indicated, but often fail to go to completion, resulting intremendous chemical heterogeneity in sulfation pattern in heparin. Theobservation that addition of exogenous heparin partially rescues onlythe K183A/K188A mutant (predicted to coordinate 6-O-sulfate) and not theN89A/K198A mutant (predicted to coordinate 2-O-sulfate), is probably dueto the fact that the heparin sub-fraction containing 2-O-sulfate is moreabundant than the sub-fraction containing 6-O-sulfate.

The heterogeneity in sulfation pattern is even more profound in heparansulfate moieties of cell surface HSPG, which are thought to cooperatewith FGFs to induce FGFR dimerization and activation. The requirementfor a specific sulfation motif in heparan sulfate for optimal FGF4action may be a mechanism to fine tune FGF4-FGFR interactions and torestrict FGF4 signaling to a specific set of cells in a specific tissueduring various stages of embryonic development, where spatial andtemporal regulation by FGF is critically required.

Implications for tile general mode of FGF-FGFR binding. Those skilled inthe art will note that some of the data presented here is not consistentwith the model of FGF1-FGFR2 binding described in the recently publishedcrystal structure of a ternary FGF1-FGFR2-heparin complex (Pellegrini etal., Nature 2000, 407:1029-1034). In the novel structure presented inPellegrini, the FGFR-invariant Pro-253 located in the D2-D3 linker, isfound in a cis configuration, while in all previously reported binaryFGF-FGFR structures Pro253 is found only in a trans configuration(Plotnikov et al., Cell 1999, 98:641-650; Plotnikov et al., Cell 2000,101:413-424; Stauber et al., Proc. Natl. Acad. Sci. U.S.A. 2000,97:49-54). Consequently, relative to its position in all binary FGF-FGFRstructures, the receptor D3 in the ternary FGF1-FGFR2-heparin structureis swiveled around the linker region by more than 160°. This creates acompletely different set of interactions at the FGF-D3 interface.Pellegrini et al. (Nature 2000, 407:1029-1034) propose that this D3rotation is caused by a heparin-mediated trans to cis isomerization ofPro253 in the D2-D3 linker region. However, the mutagenesis datapresented here do not support this hypothesis. Based on this ternaryFGF1-FGFR2-heparin structure (Pellegrini et al., supra), neither F129nor F151 in FGF4 are predicted to make any contacts with D3 (FIG. 6).Thus, the drastically reduced mitogenic capacity of the F129A and-F151Amutants is in disagreement with the mode of FGF-FGFR binding describedby those authors (i.e., by Pellegrini et al., supra) Surprisingly, thedata presented here suggested that the cis isomerization of Pro253observed in the FGF1-FGFR2-heparin structure (Pellegrini et al., supra)is probably the result of partial refolding of FGFR2.

The present invention is therefore based on a different model ofFGF-FGFR binding, presented supra, in which interaction of FGF with theFGFR D2 domain provides the primary FGF-FGFR binding surface (FIG.7A-B), and heparin facilitates the formation of an FGF-D3 interface bystabilizing the FGF-D2 interaction (FIG. 9A-B). This model may explainthe exclusively heparin-dependent binding of FGF1 to an in vitrorefolded FGFR2 described by Pellegrini et al. (supra). As discussedabove, it is likely that the FGFR2 used by these authors was notproperly refolded and consequently D3 is in a different position thanthe one observed in the previously reported FGF-FGFR crystal structures.Despite the lack of sufficient contact between FGF1 and FGFR2 D3, theFGF1-FGFR2 complex could still be captured in the presence of heparin asevident from the crystal structure (Pellegrini et al., supra).

The data presented here show that FGF4 adopts a typical β-trefoil foldsimilar to that adopted by other FGFs (see, e.g., Osslund et al.,Protein Sci. 1998, 7:1681-1690; Plotnikov et al., J. Biol. Chem. 2001,276:4322-4329; Zhu et al., J. Biol. Chem. 1995, 270:21869-21874). Theternary FGF4-FGFR1-heparin model constructed, above, by superimposingFGF4 onto FGF2 in the FGF2-FGFR1-heparin structure assistsidentification of several key residues in FGF4 involved in receptor andheparin binding. Substitution of several of these residues with alanineproduces FGF4 molecules with reduced receptor binding and mitogenicpotential, which may, at least in certain cases, be partially reversedby excess soluble heparin. Significantly, the modeling and mutagenesisdata presented here show that FGF4 interacts with the βC′-βE loop inFGFR D3 and provide a molecular basis for why FGF4, like FGF2 but unlikeFGF1, can discriminate between the IIIc and IIIb splice variants ofFGFRs for binding. Based on these findings, a skilled artisan mayreadily identify specific FGF-FGFR interactions and, from these, designnovel, variant FGF molecules with increased or altered bindingspecificity.

7. References Cited

Numerous references, including patents, patent applications and variouspublications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entirety andto the same extent as if each reference was individually incorporated byreference.

8. Appendix A: One and Three Letter Designations for Amino Acids

Alanine Ala A Isoleucine Ile I Arginine Arg R Leucine Leu L AsparagineAsn N Lysine Lys K Aspartic acid Asp D Methionine Met M Cysteine Cys CPhenylalanine Phe F Glutamine Gln Q Proline Pro P Glutamic acid Glu ESerine Ser S Glycine Gly G Threonine Thr T Histidine His H TryptophanTrp W Tyrosine Tyr Y Valine Val V

9. Appendix B: Crystal Structure Coordinates for FGF4 (SEQ ID NO: 20)

REMARK coordinates from restrained individual B-factor refinement REMARKrefinement resolution: 30–1.8 A REMARK starting r = 0.1944 free_r =0.2070 REMARK final r = 0.1943 free_r = 0.2067 REMARK B rmsd for bondedmainchain atoms = 0.695 target = 1.5 REMARK B rmsd for bonded sidechainatoms = 1.228 target = 2.0 REMARK B rmsd for angle mainchain atoms =1.094 target = 2.0 REMARK B rmsd for angle sidechain atoms = 1.939target = 2.5 REMARK wa = 0.911288 REMARK rweight = 0.235168 REMARKtarget = mlf steps = 30 REMARK sg = P2(1)2(1)2 (1) a = 40.367 b = 53.295c = 56.231 alpha = 90 beta = 90 gamma = 90 REMARK parameter file 1:CNS_TOPPAR: protein_rep.param REMARK parameter file 2: CNS_TOPPAR:dna-rna.param REMARK parameter file 3: CNS_TOPPAR: water_rep.paramREMARK parameter file 4: CNS_TOPPAR: ion.param REMARK molecularstructure file: fgf4_9.mtf REMARK input coordinates: fgf4_9X.pdb REMARKreflection file = fgf4.hklt REMARK ncs = none REMARK B-correctionresolution: 6.0–1.8 REMARK initial B-factor correction applied to fobs:REMARK B11 = −3.650 B22 = 0.683 B33 = 2.967 REMARK B12 = 0.000 B13 =0.000 B23 = 0.000 REMARK B-factor correction applied to coordinate arrayB: 0.028 REMARK bulk solvent: density level = 0.570684 e/A{circumflexover ( )}3, B-factor = 74.852 A{circumflex over ( )}2 REMARK reflectionswith |Fobs|/sigma_F > 0.0 rejected REMARK reflections with |Fobs| >10000 * rms(Fobs) rejected REMARK theoretical total number of refl. inresol. range: 11739 (100.0%) REMARK number of unobserved reflections (noentry or |F| = 0):  251 (2.1%) REMARK number of reflections rejected:  0 (0.0%) REMARK total number of reflections used: 11488 (97.9%) REMARKnumber of reflections in working set: 10894 (92.8%) REMARK number ofreflections in test set:  594 (5.1%) CRYST1 40.367 53.295 56.231 90.0090.00 90.00 P 21 21 21 REMARK FILENAME = “fgf4_9XB.pdb” REMARK DATE:25-Jan-00 22:33:40 created by user: mohammad REMARK VERSION: 0.5 ATOM 1C GLY 79 15.981 20.788 35.818 1.00 14.10 ATOM 2 O GLY 79 15.510 20.61934.693 1.00 15.42 ATOM 3 N GLY 79 13.808 21.802 36.441 1.00 15.71 ATOM 4CA GLY 79 15.211 21.566 36.865 1.00 14.54 ATOM 5 N ILE 80 17.165 20.30836.178 1.00 12.57 ATOM 6 CA ILE 80 17.971 19.552 35.234 1.00 11.35 ATOM7 CB ILE 80 19.443 19.416 35.728 1.00 11.22 ATOM 8 CG2 ILE 80 20.08920.790 35.818 1.00 10.86 ATOM 9 CG1 ILE 80 19.497 18.729 37.096 1.0011.55 ATOM 10 CD1 ILE 80 19.441 17.210 37.040 1.00 12.63 ATOM 11 C ILE80 17.393 18.163 35.008 1.00 10.56 ATOM 12 O ILE 80 16.667 17.637 35.8521.00 9.79 ATOM 13 N LYS 81 17.700 17.589 33.849 1.00 9.65 ATOM 14 CA LYS81 17.268 16.239 33.506 1.00 9.81 ATOM 15 CB LYS 81 16.448 16.227 32.2161.00 10.72 ATOM 16 CG LYS 81 14.969 16.523 32.406 1.00 12.85 ATOM 17 CDLYS 81 14.737 17.940 32.867 1.00 14.94 ATOM 18 CE LYS 81 13.254 18.22733.041 1.00 15.71 ATOM 19 NZ LYS 81 13.025 19.662 33.354 1.00 17.00 ATOM20 C LYS 81 18.548 15.446 33.304 1.00 9.37 ATOM 21 O LYS 81 19.45715.896 32.613 1.00 9.66 ATOM 22 N ARG 82 18.631 14.271 33.913 1.00 9.22ATOM 23 CA ARG 82 19.829 13.462 33.785 1.00 9.59 ATOM 24 CB ARG 8219.895 12.453 34.932 1.00 11.59 ATOM 25 CG ARG 82 19.818 13.111 36.3021.00 14.57 ATOM 26 CD ARG 82 20.077 12.130 37.423 1.00 17.30 ATOM 27 NEARG 82 21.465 11.682 37.440 1.00 18.57 ATOM 28 CZ ARG 82 21.982 10.90538.384 1.00 20.23 ATOM 29 NH1 ARG 82 21.222 10.491 39.392 1.00 21.72ATOM 30 NH2 ARG 82 23.258 10.545 38.325 1.00 20.18 ATOM 31 C ARG 8219.904 12.741 32.447 1.00 9.37 ATOM 32 O ARG 82 18.891 12.279 31.9201.00 8.91 ATOM 33 N LEU 83 21.112 12.674 31.895 1.00 8.33 ATOM 34 CA LEU83 21.367 11.987 30.634 1.00 9.07 ATOM 35 CB LEU 83 22.237 12.843 29.7081.00 9.05 ATOM 36 CG LEU 83 21.697 14.220 29.331 1.00 9.81 ATOM 37 CD1LEU 83 22.633 14.866 28.318 1.00 9.99 ATOM 38 CD2 LEU 83 20.301 14.08828.750 1.00 9.81 ATOM 39 C LEU 83 22.133 10.747 31.055 1.00 10.07 ATOM40 O LEU 83 23.229 10.847 31.607 1.00 9.78 ATOM 41 N ARG 84 21.561 9.57630.812 1.00 9.78 ATOM 42 CA ARG 84 22.220 8.354 31.234 1.00 11.27 ATOM43 CB ARG 84 21.635 7.907 32.573 1.00 13.30 ATOM 44 CG ARG 84 21.6249.002 33.617 1.00 16.93 ATOM 45 CD ARG 84 22.305 8.543 34.881 1.00 18.68ATOM 46 NE ARG 84 21.347 8.127 35.893 1.00 21.27 ATOM 47 CZ ARG 8421.689 7.665 37.088 1.00 21.80 ATOM 48 NH1 ARG 84 22.971 7.558 37.4151.00 23.39 ATOM 49 NH2 ARG 84 20.752 7.322 37.959 1.00 23.50 ATOM 50 CARG 84 22.098 7.212 30.253 1.00 9.55 ATOM 51 O ARG 84 21.488 7.33529.199 1.00 8.79 ATOM 52 N ARG 85 22.712 6.095 30.617 1.00 9.72 ATOM 53CA ARG 85 22.632 4.886 29.823 1.00 9.53 ATOM 54 CB ARG 85 24.026 4.41029.393 1.00 11.00 ATOM 55 CG ARG 85 24.690 5.336 28.390 1.00 13.65 ATOM56 CD ARG 85 25.824 4.654 27.640 1.00 15.37 ATOM 57 NE ARG 85 26.2695.472 26.517 1.00 18.74 ATOM 58 CZ ARG 85 26.907 6.630 26.638 1.00 19.28ATOM 59 NH1 ARG 85 27.193 7.113 27.837 1.00 20.48 ATOM 60 NH2 ARG 8527.236 7.313 25.552 1.00 21.17 ATOM 61 C ARG 85 21.981 3.873 30.751 1.009.35 ATOM 62 O ARG 85 22.216 3.891 31.962 1.00 9.90 ATOM 63 N LEU 8621.128 3.024 30.194 1.00 8.17 ATOM 64 CA LEU 86 20.452 2.000 30.978 1.007.93 ATOM 65 CB LEU 86 18.945 2.045 30.711 1.00 7.25 ATOM 66 CG LEU 8618.309 3.365 31.168 1.00 7.91 ATOM 67 CD1 LEU 86 16.839 3.414 30.7691.00 7.67 ATOM 68 CD2 LEU 86 18.448 3.496 32.681 1.00 7.54 ATOM 69 C LEU86 21.061 0.673 30.552 1.00 8.05 ATOM 70 O LEU 86 20.845 0.199 29.4371.00 7.14 ATOM 71 N TYR 87 21.839 0.093 31.461 1.00 8.27 ATOM 72 CA TYR87 22.559 −1.150 31.218 1.00 9.30 ATOM 73 CB TYR 87 23.936 −1.046 31.8771.00 10.81 ATOM 74 CG TYR 87 24.805 −2.278 31.762 1.00 13.81 ATOM 75 CD1TYR 87 24.886 −3.200 32.808 1.00 14.83 ATOM 76 CE1 TYR 87 25.721 −4.31732.720 1.00 16.91 ATOM 77 CD2 TYR 87 25.576 −2.503 30.621 1.00 15.00ATOM 78 CE2 TYR 87 26.412 −3.615 30.522 1.00 16.14 ATOM 79 CZ TYR 8726.481 −4.514 31.575 1.00 16.64 ATOM 80 OH TYR 87 27.319 −5.601 31.4831.00 19.14 ATOM 81 C TYR 87 21.850 −2.412 31.689 1.00 8.55 ATOM 82 O TYR87 21.529 −2.552 32.865 1.00 7.71 ATOM 83 N CYS 88 21.613 −3.327 30.7541.00 8.66 ATOM 84 CA CYS 88 20.969 −4.598 31.058 1.00 8.99 ATOM 85 CBCYS 88 19.961 −4.956 29.964 1.00 9.37 ATOM 86 SG CYS 88 18.868 −6.33230.382 1.00 10.25 ATOM 87 C CYS 88 22.094 −5.630 31.102 1.00 9.81 ATOM88 O CYS 88 22.782 −5.850 30.105 1.00 9.57 ATOM 89 N ASN 89 22.278−6.262 32.256 1.00 10.42 ATOM 90 CA ASN 89 23.353 −7.228 32.416 1.0011.44 ATOM 91 CB ASN 89 23.800 −7.259 33.882 1.00 12.56 ATOM 92 CG ASN89 25.097 −8.023 34.078 1.00 14.22 ATOM 93 OD1 ASN 89 25.975 −7.99733.217 1.00 14.10 ATOM 94 ND2 ASN 89 25.229 −8.693 35.219 1.00 14.82ATOM 95 C ASN 89 23.021 −8.639 31.935 1.00 11.60 ATOM 96 O ASN 89 23.029−9.589 32.717 1.00 12.87 ATOM 97 N VAL 90 22.724 −8.762 30.644 1.0011.13 ATOM 98 CA VAL 90 22.414 −10.052 30.030 1.00 11.34 ATOM 99 CB VAL90 20.936 −10.130 29.553 1.00 11.83 ATOM 100 CG1 VAL 90 19.998 −10.05830.752 1.00 12.67 ATOM 101 CG2 VAL 90 20.636 −9.008 28.566 1.00 11.70ATOM 102 C VAL 90 23.344 −10.214 28.830 1.00 10.91 ATOM 103 O VAL 9023.743 −9.227 28.214 1.00 10.94 ATOM 104 N GLY 91 23.689 −11.451 28.4901.00 11.30 ATOM 105 CA GLY 91 24.596 −11.655 27.377 1.00 11.22 ATOM 106C GLY 91 25.905 −10.960 27.710 1.00 11.54 ATOM 107 O GLY 91 26.358−11.021 28.851 1.00 11.96 ATOM 108 N ILE 92 26.515 −10.295 26.735 1.0011.95 ATOM 109 CA ILE 92 27.770 −9.590 26.983 1.00 12.07 ATOM 110 CB ILE92 28.553 −9.333 25.673 1.00 12.90 ATOM 111 CG2 ILE 92 29.022 −10.65325.075 1.00 13.76 ATOM 112 CG1 ILE 92 27.679 −8.557 24.684 1.00 12.69ATOM 113 CD1 ILE 92 28.396 −8.175 23.403 1.00 13.90 ATOM 114 C ILE 9227.526 −8.241 27.661 1.00 11.76 ATOM 115 O ILE 92 28.470 −7.516 27.9741.00 12.10 ATOM 116 N GLY 93 26.258 −7.912 27.888 1.00 11.33 ATOM 117 CAGLY 93 25.921 −6.643 28.513 1.00 10.40 ATOM 118 C GLY 93 25.518 −5.63427.454 1.00 10.14 ATOM 119 O GLY 93 26.279 −5.386 26.514 1.00 9.39 ATOM120 N PHE 94 24.330 −5.048 27.587 1.00 9.60 ATOM 121 CA PHE 94 23.868−4.081 26.591 1.00 9.48 ATOM 122 CB PHE 94 22.881 −4.738 25.620 1.0010.07 ATOM 123 CG PHE 94 23.416 −5.959 24.932 1.00 11.72 ATOM 124 CD1PHE 94 23.281 −7.215 25.512 1.00 11.80 ATOM 125 CD2 PHE 94 24.052 −5.85123.699 1.00 12.37 ATOM 126 CE1 PHE 94 23.768 −8.350 24.872 1.00 12.80ATOM 127 CE2 PHE 94 24.544 −6.977 23.052 1.00 13.09 ATOM 128 CZ PHE 9424.402 −8.229 23.637 1.00 13.50 ATOM 129 C PHE 94 23.196 −2.837 27.1621.00 9.07 ATOM 130 O PHE 94 22.530 −2.898 28.194 1.00 9.31 ATOM 131 NHIS 95 23.372 −1.712 26.473 1.00 9.61 ATOM 132 CA HIS 95 22.739 −0.45426.868 1.00 8.83 ATOM 133 CB HIS 95 23.667 0.755 26.670 1.00 9.80 ATOM134 CG HIS 95 24.991 0.636 27.356 1.00 11.64 ATOM 135 CD2 HIS 95 25.3740.950 28.614 1.00 11.84 ATOM 136 ND1 HIS 95 26.112 0.151 26.722 1.0012.48 ATOM 137 CE1 HIS 95 27.133 0.170 27.559 1.00 12.86 ATOM 138 NE2HIS 95 26.712 0.650 28.717 1.00 13.05 ATOM 139 C HIS 95 21.544 −0.27025.940 1.00 8.68 ATOM 140 O HIS 95 21.663 −0.449 24.728 1.00 8.37 ATOM141 N LEU 96 20.397 0.086 26.505 1.00 7.77 ATOM 142 CA LEU 96 19.1980.312 25.711 1.00 7.43 ATOM 143 CB LEU 96 18.028 0.661 26.634 1.00 7.74ATOM 144 CG LEU 96 16.658 0.911 26.000 1.00 7.64 ATOM 145 CD1 LEU 9616.145 −0.363 25.333 1.00 8.64 ATOM 146 CD2 LEU 96 15.689 1.369 27.0831.00 7.70 ATOM 147 C LEU 96 19.485 1.479 24.763 1.00 7.66 ATOM 148 O LEU96 20.047 2.492 25.177 1.00 6.82 ATOM 149 N GLN 97 19.120 1.341 23.4931.00 7.35 ATOM 150 CA GLN 97 19.369 2.430 22.556 1.00 8.13 ATOM 151 CBGLN 97 20.675 2.190 21.787 1.00 9.41 ATOM 152 CG GLN 97 20.610 1.13020.698 1.00 11.56 ATOM 153 CD GLN 97 21.901 1.049 19.892 1.00 13.36 ATOM154 OE1 GLN 97 22.723 1.967 19.919 1.00 14.09 ATOM 155 NE2 GLN 97 22.076−0.045 19.159 1.00 13.35 ATOM 156 C GLN 97 18.231 2.650 21.571 1.00 7.87ATOM 157 O GLN 97 17.467 1.735 21.265 1.00 6.47 ATOM 158 N ALA 98 18.1193.884 21.091 1.00 7.19 ATOM 159 CA ALA 98 17.094 4.242 20.122 1.00 7.41ATOM 160 CB ALA 98 16.259 5.399 20.650 1.00 6.51 ATOM 161 C ALA 9817.817 4.642 18.839 1.00 7.88 ATOM 162 O ALA 98 18.651 5.549 18.846 1.007.94 ATOM 163 N LEU 99 17.504 3.960 17.743 1.00 8.72 ATOM 164 CA LEU 9918.149 4.237 16.465 1.00 9.77 ATOM 165 CB LEU 99 18.245 2.946 15.6511.00 10.44 ATOM 166 CG LEU 99 19.040 1.816 16.318 1.00 11.58 ATOM 167CD1 LEU 99 19.132 0.639 15.367 1.00 13.27 ATOM 168 CD2 LEU 99 20.4332.299 16.678 1.00 11.97 ATOM 169 C LEU 99 17.441 5.321 15.657 1.00 9.49ATOM 170 O LEU 99 16.262 5.599 15.871 1.00 10.13 ATOM 171 N PRO 10018.159 5.943 14.709 1.00 9.88 ATOM 172 CD PRO 100 19.583 5.707 14.4031.00 10.62 ATOM 173 CA PRO 100 17.615 7.006 13.858 1.00 10.16 ATOM 174CB PRO 100 18.847 7.512 13.105 1.00 10.38 ATOM 175 CG PRO 100 19.7076.290 13.012 1.00 11.95 ATOM 176 C PRO 100 16.482 6.603 12.921 1.00 9.96ATOM 177 O PRO 100 15.811 7.471 12.358 1.00 9.98 ATOM 178 N ASP 10116.261 5.301 12.743 1.00 9.35 ATOM 179 CA ASP 101 15.175 4.862 11.8741.00 8.93 ATOM 180 CB ASP 101 15.613 3.683 10.988 1.00 8.94 ATOM 181 CGASP 101 16.058 2.464 11.779 1.00 9.32 ATOM 182 OD1 ASP 101 16.005 2.47813.028 1.00 7.56 ATOM 183 OD2 ASP 101 16.467 1.474 11.132 1.00 9.79 ATOM184 C ASP 101 13.923 4.499 12.668 1.00 8.49 ATOM 185 O ASP 101 12.9144.074 12.098 1.00 7.97 ATOM 186 N GLY 102 13.992 4.681 13.985 1.00 7.41ATOM 187 CA GLY 102 12.851 4.386 14.835 1.00 6.68 ATOM 188 C GLY 10212.906 3.057 15.562 1.00 6.54 ATOM 189 O GLY 102 12.011 2.740 16.3441.00 6.36 ATOM 190 N ARG 103 13.946 2.268 15.316 1.00 5.93 ATOM 191 CAARG 103 14.068 0.982 15.987 1.00 6.49 ATOM 192 CB ARG 103 14.961 0.03415.174 1.00 7.14 ATOM 193 CG ARG 103 14.338 −0.417 13.854 1.00 8.91 ATOM194 CD ARG 103 15.206 −1.444 13.124 1.00 11.34 ATOM 195 NE ARG 10315.547 −2.579 13.979 1.00 13.84 ATOM 196 CZ ARG 103 16.740 −2.764 14.5401.00 15.57 ATOM 197 NH1 ARG 103 16.956 −3.823 15.309 1.00 16.83 ATOM 198NH2 ARG 103 17.725 −1.901 14.317 1.00 15.44 ATOM 199 C ARG 103 14.6321.154 17.394 1.00 6.57 ATOM 200 O ARG 103 15.241 2.175 17.713 1.00 7.34ATOM 201 N ILE 104 14.404 0.149 18.230 1.00 5.76 ATOM 202 CA ILE 10414.883 0.138 19.606 1.00 6.17 ATOM 203 CB ILE 104 13.705 0.097 20.6211.00 5.44 ATOM 204 CG2 ILE 104 14.243 0.072 22.047 1.00 5.86 ATOM 205CG1 ILE 104 12.780 1.306 20.427 1.00 5.60 ATOM 206 CD1 ILE 104 13.4242.652 20.740 1.00 6.40 ATOM 207 C ILE 104 15.704 −1.146 19.752 1.00 6.60ATOM 208 O ILE 104 15.334 −2.190 19.208 1.00 6.24 ATOM 209 N GLY 10516.820 −1.071 20.468 1.00 6.98 ATOM 210 CA GLY 105 17.641 −2.255 20.6451.00 8.04 ATOM 211 C GLY 105 18.680 −2.100 21.737 1.00 8.60 ATOM 212 OGLY 105 18.540 −1.255 22.620 1.00 8.84 ATOM 213 N GLY 106 19.723 −2.92321.674 1.00 8.72 ATOM 214 CA GLY 106 20.779 −2.861 22.667 1.00 9.01 ATOM215 C GLY 106 22.146 −2.743 22.021 1.00 8.89 ATOM 216 O GLY 106 22.356−3.219 20.905 1.00 8.84 ATOM 217 N ALA 107 23.072 −2.092 22.715 1.008.61 ATOM 218 CA ALA 107 24.432 −1.920 22.213 1.00 9.67 ATOM 219 CB ALA107 24.604 −0.528 21.620 1.00 10.06 ATOM 220 C ALA 107 25.409 −2.12423.364 1.00 10.28 ATOM 221 O ALA 107 25.273 −1.508 24.418 1.00 9.28 ATOM222 N HIS 108 26.391 −2.995 23.161 1.00 11.70 ATOM 223 CA HIS 108 27.374−3.270 24.198 1.00 12.04 ATOM 224 CB HIS 108 28.253 −4.455 23.786 1.0013.11 ATOM 225 CG HIS 108 29.307 −4.801 24.792 1.00 14.15 ATOM 226 CD2HIS 108 30.657 −4.798 24.702 1.00 14.78 ATOM 227 ND1 HIS 108 29.008−5.191 26.079 1.00 14.21 ATOM 228 CE1 HIS 108 30.129 −5.412 26.741 1.0015.02 ATOM 229 NE2 HIS 108 31.146 −5.181 25.927 1.00 16.19 ATOM 230 CHIS 108 28.244 −2.048 24.480 1.00 12.52 ATOM 231 O HIS 108 28.554 −1.75225.633 1.00 12.77 ATOM 232 N ALA 109 28.631 −1.334 23.429 1.00 13.09ATOM 233 CA ALA 109 29.470 −0.150 23.587 1.00 13.18 ATOM 234 CB ALA 10930.199 0.147 22.280 1.00 14.30 ATOM 235 C ALA 109 28.654 1.067 24.0161.00 13.45 ATOM 236 O ALA 109 27.423 1.040 23.985 1.00 12.92 ATOM 237 NASP 110 29.344 2.127 24.427 1.00 13.54 ATOM 238 CA ASP 110 28.674 3.35524.841 1.00 14.78 ATOM 239 CB ASP 110 29.548 4.164 25.807 1.00 16.93ATOM 240 CG ASP 110 29.747 3.470 27.143 1.00 19.56 ATOM 241 OD1 ASP 11028.758 2.968 27.715 1.00 20.96 ATOM 242 OD2 ASP 110 30.897 3.442 27.6311.00 21.98 ATOM 243 C ASP 110 28.375 4.190 23.599 1.00 13.85 ATOM 244 OASP 110 28.956 5.257 23.399 1.00 13.85 ATOM 245 N THR 111 27.465 3.68722.773 1.00 12.59 ATOM 246 CA THR 111 27.066 4.351 21.532 1.00 11.75ATOM 247 CB THR 111 26.120 3.456 20.709 1.00 11.78 ATOM 248 OG1 THR 11124.926 3.205 21.464 1.00 11.42 ATOM 249 CG2 THR 111 26.783 2.133 20.3781.00 11.72 ATOM 250 C THR 111 26.340 5.666 21.779 1.00 11.02 ATOM 251 OTHR 111 25.736 5.864 22.831 1.00 10.14 ATOM 252 N ARG 112 26.397 6.56520.799 1.00 10.53 ATOM 253 CA ARG 112 25.715 7.845 20.918 1.00 10.82ATOM 254 CB ARG 112 25.941 8.701 19.669 1.00 12.11 ATOM 255 CG ARG 11227.380 9.110 19.432 1.00 14.73 ATOM 256 CD ARG 112 27.470 9.990 18.1981.00 17.34 ATOM 257 NE ARG 112 28.842 10.372 17.886 1.00 19.56 ATOM 258CZ ARG 112 29.169 11.260 16.955 1.00 20.19 ATOM 259 NH1 ARG 112 28.21811.857 16.248 1.00 21.18 ATOM 260 NH2 ARG 112 30.443 11.552 16.729 1.0021.74 ATOM 261 C ARG 112 24.220 7.604 21.083 1.00 10.26 ATOM 262 O ARG112 23.562 8.275 21.874 1.00 10.16 ATOM 263 N ASP 113 23.694 6.63520.335 1.00 9.55 ATOM 264 CA ASP 113 22.270 6.319 20.385 1.00 9.39 ATOM265 CB ASP 113 21.875 5.410 19.214 1.00 9.84 ATOM 266 CG ASP 113 21.9936.103 17.867 1.00 11.80 ATOM 267 OD1 ASP 113 22.074 7.351 17.838 1.0012.79 ATOM 268 OD2 ASP 113 21.991 5.402 16.833 1.00 11.25 ATOM 269 C ASP113 21.802 5.686 21.690 1.00 8.59 ATOM 270 O ASP 113 20.604 5.473 21.8761.00 7.24 ATOM 271 N SER 114 22.730 5.386 22.595 1.00 8.92 ATOM 272 CASER 114 22.343 4.794 23.871 1.00 8.63 ATOM 273 CB SER 114 23.294 3.65024.256 1.00 9.68 ATOM 274 OG SER 114 24.625 4.100 24.427 1.00 10.49 ATOM275 C SER 114 22.286 5.836 24.989 1.00 8.56 ATOM 276 O SER 114 22.0425.495 26.145 1.00 8.23 ATOM 277 N LEU 115 22.520 7.103 24.651 1.00 7.61ATOM 278 CA LEU 115 22.445 8.164 25.652 1.00 7.24 ATOM 279 CB LEU 11523.260 9.386 25.230 1.00 8.19 ATOM 280 CG LEU 115 23.344 10.488 26.2921.00 8.61 ATOM 281 CD1 LEU 115 24.133 9.981 27.504 1.00 9.28 ATOM 282CD2 LEU 115 24.015 11.717 25.703 1.00 9.34 ATOM 283 C LEU 115 20.9728.534 25.745 1.00 6.88 ATOM 284 O LEU 115 20.374 8.976 24.762 1.00 7.36ATOM 285 N LEU 116 20.389 8.353 26.923 1.00 6.00 ATOM 286 CA LEU 11618.968 8.621 27.108 1.00 6.40 ATOM 287 CB LEU 116 18.266 7.325 27.5281.00 6.05 ATOM 288 CG LEU 116 18.557 6.068 26.702 1.00 5.86 ATOM 289 CD1LEU 116 17.940 4.851 27.392 1.00 6.33 ATOM 290 CD2 LEU 116 17.991 6.23125.304 1.00 6.19 ATOM 291 C LEU 116 18.663 9.703 28.137 1.00 7.23 ATOM292 O LEU 116 19.225 9.710 29.230 1.00 8.16 ATOM 293 N GLU 117 17.77110.621 27.785 1.00 7.97 ATOM 294 CA GLU 117 17.384 11.669 28.723 1.008.82 ATOM 295 CB GLU 117 17.043 12.971 27.990 1.00 10.69 ATOM 296 CG GLU117 16.520 14.065 28.914 1.00 12.86 ATOM 297 CD GLU 117 16.478 15.43328.258 1.00 16.05 ATOM 298 OE1 GLU 117 15.991 16.386 28.903 1.00 17.79ATOM 299 OE2 GLU 117 16.937 15.562 27.105 1.00 17.80 ATOM 300 C GLU 11716.164 11.174 29.490 1.00 9.20 ATOM 301 O GLU 117 15.122 10.891 28.8991.00 8.75 ATOM 302 N LEU 118 16.305 11.045 30.804 1.00 8.92 ATOM 303 CALEU 118 15.204 10.592 31.642 1.00 9.72 ATOM 304 CB LEU 118 15.728 9.72232.786 1.00 11.39 ATOM 305 CG LEU 118 16.532 8.480 32.394 1.00 12.95ATOM 306 CD1 LEU 118 16.853 7.667 33.643 1.00 14.11 ATOM 307 CD2 LEU 11815.743 7.643 31.409 1.00 14.39 ATOM 308 C LEU 118 14.508 11.826 32.2001.00 10.39 ATOM 309 O LEU 118 15.103 12.596 32.955 1.00 9.73 ATOM 310 NSER 119 13.251 12.024 31.819 1.00 10.03 ATOM 311 CA SER 119 12.51013.187 32.284 1.00 10.69 ATOM 312 CB SER 119 12.014 14.005 31.091 1.0011.30 ATOM 313 OG SER 119 13.080 14.335 30.217 1.00 11.39 ATOM 314 C SER119 11.321 12.770 33.132 1.00 10.86 ATOM 315 O SER 119 10.503 11.95932.706 1.00 10.01 ATOM 316 N PRO 120 11.213 13.315 34.350 1.00 11.88ATOM 317 CD PRO 120 12.109 14.265 35.038 1.00 12.61 ATOM 318 CA PRO 12010.081 12.950 35.202 1.00 12.25 ATOM 319 CB PRO 120 10.441 13.581 36.5471.00 13.23 ATOM 320 CG PRO 120 11.220 14.799 36.145 1.00 14.45 ATOM 321C PRO 120 8.782 13.505 34.623 1.00 12.69 ATOM 322 O PRO 120 8.730 14.64834.166 1.00 12.96 ATOM 323 N VAL 121 7.742 12.682 34.627 1.00 12.46 ATOM324 CA VAL 121 6.440 13.088 34.118 1.00 12.87 ATOM 325 CB VAL 121 5.79711.948 33.306 1.00 12.58 ATOM 326 CG1 VAL 121 4.378 12.318 32.902 1.0011.86 ATOM 327 CG2 VAL 121 6.642 11.666 32.074 1.00 13.10 ATOM 328 C VAL121 5.586 13.418 35.332 1.00 12.84 ATOM 329 O VAL 121 4.902 14.44035.378 1.00 13.81 ATOM 330 N GLU 122 5.654 12.532 36.316 1.00 12.83 ATOM331 CA GLU 122 4.949 12.667 37.583 1.00 12.89 ATOM 332 CB GLU 122 3.48812.222 37.457 1.00 13.88 ATOM 333 CG GLU 122 3.266 10.931 36.691 1.0015.79 ATOM 334 CD GLU 122 1.805 10.506 36.688 1.00 17.76 ATOM 335 OE1GLU 122 1.385 9.812 35.741 1.00 18.31 ATOM 336 OE2 GLU 122 1.078 10.85637.642 1.00 19.25 ATOM 337 C GLU 122 5.705 11.759 38.538 1.00 12.49 ATOM338 O GLU 122 6.672 11.114 38.135 1.00 12.27 ATOM 339 N ARG 123 5.28811.701 39.795 1.00 11.54 ATOM 340 CA ARG 123 5.998 10.854 40.739 1.0010.92 ATOM 341 CB ARG 123 5.344 10.896 42.120 1.00 11.82 ATOM 342 CG ARG123 5.972 9.885 43.059 1.00 12.96 ATOM 343 CD ARG 123 5.602 10.11244.501 1.00 14.90 ATOM 344 NE ARG 123 6.104 9.014 45.318 1.00 14.69 ATOM345 CZ ARG 123 6.118 9.015 46.644 1.00 14.66 ATOM 346 NH1 ARG 123 5.65810.067 47.309 1.00 15.83 ATOM 347 NH2 ARG 123 6.579 7.963 47.302 1.0013.18 ATOM 348 C ARG 123 6.109 9.399 40.288 1.00 9.45 ATOM 349 O ARG 1235.101 8.744 40.018 1.00 9.21 ATOM 350 N GLY 124 7.345 8.909 40.217 1.008.93 ATOM 351 CA GLY 124 7.601 7.530 39.828 1.00 8.60 ATOM 352 C GLY 1247.471 7.208 38.349 1.00 7.77 ATOM 353 O GLY 124 7.656 6.060 37.947 1.007.28 ATOM 354 N VAL 125 7.169 8.215 37.537 1.00 7.41 ATOM 355 CA VAL 1256.989 8.018 36.101 1.00 7.25 ATOM 356 CB VAL 125 5.546 8.377 35.687 1.006.87 ATOM 357 CG1 VAL 125 5.339 8.101 34.212 1.00 7.15 ATOM 358 CG2 VAL125 4.552 7.585 36.530 1.00 7.51 ATOM 359 C VAL 125 7.955 8.874 35.2911.00 7.01 ATOM 360 O VAL 125 8.153 10.052 35.593 1.00 7.46 ATOM 361 NVAL 126 8.539 8.286 34.249 1.00 6.88 ATOM 362 CA VAL 126 9.493 8.99933.403 1.00 6.56 ATOM 363 CB VAL 126 10.942 8.553 33.703 1.00 6.83 ATOM364 CG1 VAL 126 11.302 8.836 35.148 1.00 6.87 ATOM 365 CG2 VAL 12611.087 7.064 33.401 1.00 7.21 ATOM 366 C VAL 126 9.290 8.760 31.911 1.006.87 ATOM 367 O VAL 126 8.579 7.842 31.505 1.00 7.11 ATOM 368 N SER 1279.911 9.612 31.099 1.00 5.95 ATOM 369 CA SER 127 9.895 9.432 29.655 1.006.02 ATOM 370 CB SER 127 9.447 10.696 28.908 1.00 6.80 ATOM 371 OG SER127 10.344 11.774 29.083 1.00 7.48 ATOM 372 C SER 127 11.366 9.13029.384 1.00 6.01 ATOM 373 O SER 127 12.243 9.559 30.143 1.00 6.11 ATOM374 N ILE 128 11.636 8.375 28.330 1.00 5.10 ATOM 375 CA ILE 128 13.0037.997 27.999 1.00 5.69 ATOM 376 CB ILE 128 13.170 6.472 28.121 1.00 5.40ATOM 377 CG2 ILE 128 14.626 6.088 27.897 1.00 6.23 ATOM 378 CG1 ILE 12812.698 6.014 29.511 1.00 5.86 ATOM 379 CD1 ILE 128 12.683 4.497 29.7131.00 5.16 ATOM 380 C ILE 128 13.268 8.455 26.572 1.00 5.76 ATOM 381 OILE 128 12.789 7.849 25.615 1.00 6.10 ATOM 382 N PHE 129 14.041 9.53026.442 1.00 5.77 ATOM 383 CA PHE 129 14.324 10.126 25.141 1.00 6.64 ATOM384 CB PHE 129 14.092 11.638 25.237 1.00 7.28 ATOM 385 CG PHE 129 14.16712.355 23.920 1.00 7.76 ATOM 386 CD1 PHE 129 13.172 12.186 22.965 1.008.17 ATOM 387 CD2 PHE 129 15.227 13.214 23.643 1.00 8.14 ATOM 388 CE1PHE 129 13.227 12.867 21.752 1.00 8.24 ATOM 389 CE2 PHE 129 15.29313.897 22.435 1.00 8.09 ATOM 390 CZ PHE 129 14.291 13.725 21.488 1.008.83 ATOM 391 C PHE 129 15.722 9.859 24.588 1.00 7.07 ATOM 392 O PHE 12916.726 10.118 25.253 1.00 6.90 ATOM 393 N GLY 130 15.773 9.344 23.3631.00 7.82 ATOM 394 CA GLY 130 17.048 9.073 22.720 1.00 7.44 ATOM 395 CGLY 130 17.588 10.389 22.195 1.00 8.24 ATOM 396 O GLY 130 17.134 10.89221.170 1.00 7.64 ATOM 397 N VAL 131 18.562 10.944 22.908 1.00 8.47 ATOM398 CA VAL 131 19.164 12.226 22.561 1.00 8.95 ATOM 399 CB VAL 131 20.37112.512 23.489 1.00 10.08 ATOM 400 CG1 VAL 131 20.979 13.857 23.157 1.0012.10 ATOM 401 CG2 VAL 131 19.917 12.487 24.948 1.00 11.84 ATOM 402 CVAL 131 19.606 12.383 21.107 1.00 8.99 ATOM 403 O VAL 131 19.258 13.36420.447 1.00 8.93 ATOM 404 N ALA 132 20.367 11.416 20.606 1.00 7.92 ATOM405 CA ALA 132 20.880 11.473 19.241 1.00 8.44 ATOM 406 CB ALA 132 22.08210.529 19.103 1.00 8.07 ATOM 407 C ALA 132 19.864 11.170 18.142 1.008.83 ATOM 408 O ALA 132 19.806 11.876 17.132 1.00 9.23 ATOM 409 N SER133 19.066 10.123 18.337 1.00 8.54 ATOM 410 CA SER 133 18.076 9.72117.341 1.00 9.10 ATOM 411 CB SER 133 17.626 8.279 17.592 1.00 9.66 ATOM412 OG SER 133 16.807 8.212 18.748 1.00 12.46 ATOM 413 C SER 133 16.85010.627 17.356 1.00 9.35 ATOM 414 O SER 133 16.158 10.761 16.348 1.008.28 ATOM 415 N ARG 134 16.585 11.234 18.510 1.00 9.32 ATOM 416 CA ARG134 15.436 12.113 18.696 1.00 10.27 ATOM 417 CB ARG 134 15.380 13.17017.588 1.00 12.72 ATOM 418 CG ARG 134 16.623 14.046 17.501 1.00 16.89ATOM 419 CD ARG 134 16.855 14.803 18.799 1.00 19.81 ATOM 420 NE ARG 13418.015 15.691 18.743 1.00 23.62 ATOM 421 CZ ARG 134 18.138 16.699 17.8861.00 24.68 ATOM 422 NH1 ARG 134 17.172 16.945 17.012 1.00 25.92 ATOM 423NH2 ARG 134 19.218 17.471 17.911 1.00 25.37 ATOM 424 C ARG 134 14.12111.331 18.745 1.00 9.51 ATOM 425 O ARG 134 13.077 11.837 18.339 1.0010.33 ATOM 426 N PHE 135 14.178 10.097 19.237 1.00 8.65 ATOM 427 CA PHE135 12.980 9.269 19.374 1.00 8.38 ATOM 428 CB PHE 135 13.106 7.94018.621 1.00 8.83 ATOM 429 CG PHE 135 12.898 8.035 17.139 1.00 8.66 ATOM430 CD1 PHE 135 13.953 8.356 16.291 1.00 10.75 ATOM 431 CD2 PHE 13511.649 7.765 16.584 1.00 8.52 ATOM 432 CE1 PHE 135 13.768 8.404 14.9091.00 10.30 ATOM 433 CE2 PHE 135 11.450 7.811 15.205 1.00 8.67 ATOM 434CZ PHE 135 12.512 8.131 14.366 1.00 10.46 ATOM 435 C PHE 135 12.7688.924 20.842 1.00 7.92 ATOM 436 O PHE 135 13.730 8.669 21.564 1.00 7.39ATOM 437 N PHE 136 11.512 8.920 21.279 1.00 7.93 ATOM 438 CA PHE 13611.184 8.529 22.642 1.00 7.08 ATOM 439 CB PHE 136 9.824 9.077 23.0911.00 7.49 ATOM 440 CG PHE 136 9.838 10.515 23.482 1.00 6.98 ATOM 441 CD1PHE 136 9.518 11.501 22.560 1.00 7.11 ATOM 442 CD2 PHE 136 10.155 10.88424.785 1.00 7.49 ATOM 443 CE1 PHE 136 9.508 12.839 22.928 1.00 8.41 ATOM444 CE2 PHE 136 10.150 12.218 25.166 1.00 8.09 ATOM 445 CZ PHE 136 9.82513.200 24.233 1.00 7.87 ATOM 446 C PHE 136 11.033 7.019 22.576 1.00 7.61ATOM 447 O PHE 136 10.617 6.483 21.548 1.00 7.88 ATOM 448 N VAL 13711.378 6.331 23.654 1.00 6.58 ATOM 449 CA VAL 137 11.189 4.890 23.6911.00 6.26 ATOM 450 CB VAL 137 12.017 4.231 24.805 1.00 6.19 ATOM 451 CG1VAL 137 11.626 2.759 24.937 1.00 6.00 ATOM 452 CG2 VAL 137 13.503 4.35124.488 1.00 6.61 ATOM 453 C VAL 137 9.708 4.749 24.027 1.00 6.37 ATOM454 O VAL 137 9.213 5.417 24.937 1.00 6.96 ATOM 455 N ALA 138 8.9973.907 23.286 1.00 6.10 ATOM 456 CA ALA 138 7.568 3.707 23.522 1.00 6.34ATOM 457 CB ALA 138 6.750 4.478 22.479 1.00 6.40 ATOM 458 C ALA 1387.220 2.226 23.461 1.00 6.19 ATOM 459 O ALA 138 7.998 1.418 22.959 1.005.65 ATOM 460 N MET 139 6.053 1.866 23.984 1.00 6.77 ATOM 461 CA MET 1395.622 0.472 23.949 1.00 6.76 ATOM 462 CB MET 139 5.802 −0.193 25.3161.00 7.13 ATOM 463 CG MET 139 5.454 −1.675 25.304 1.00 8.40 ATOM 464 SDMET 139 5.687 −2.479 26.897 1.00 9.30 ATOM 465 CE MET 139 7.482 −2.49627.008 1.00 8.92 ATOM 466 C MET 139 4.161 0.391 23.529 1.00 7.49 ATOM467 O MET 139 3.318 1.120 24.055 1.00 6.81 ATOM 468 N SER 140 3.873−0.493 22.576 1.00 7.52 ATOM 469 CA SER 140 2.515 −0.679 22.068 1.008.67 ATOM 470 CB SER 140 2.557 −1.319 20.678 1.00 8.60 ATOM 471 OG SER140 2.937 −2.684 20.771 1.00 7.74 ATOM 472 C SER 140 1.696 −1.573 22.9961.00 9.77 ATOM 473 O SER 140 2.226 −2.148 23.948 1.00 9.80 ATOM 474 NSER 141 0.407 −1.702 22.701 1.00 9.71 ATOM 475 CA SER 141 −0.483 −2.53023.510 1.00 11.35 ATOM 476 CB SER 141 −1.939 −2.307 23.091 1.00 11.04ATOM 477 OG SER 141 −2.161 −2.753 21.763 1.00 12.07 ATOM 478 C SER 141−0.130 −4.010 23.375 1.00 11.94 ATOM 479 O SER 141 −0.628 −4.848 24.1311.00 12.36 ATOM 480 N LYS 142 0.724 −4.327 22.406 1.00 12.31 ATOM 481 CALYS 142 1.150 −5.705 22.186 1.00 13.17 ATOM 482 CB LYS 142 1.258 −6.00520.690 1.00 15.47 ATOM 483 CG LYS 142 −0.063 −5.947 19.946 1.00 18.63ATOM 484 CD LYS 142 0.144 −6.189 18.463 1.00 21.02 ATOM 485 CE LYS 142−1.155 −6.052 17.690 1.00 23.01 ATOM 486 NZ LYS 142 −0.927 −6.222 16.2281.00 24.37 ATOM 487 C LYS 142 2.498 −5.950 22.849 1.00 12.17 ATOM 488 OLYS 142 3.071 −7.027 22.717 1.00 12.32 ATOM 489 N GLY 143 3.000 −4.93623.550 1.00 10.43 ATOM 490 CA GLY 143 4.270 −5.061 24.245 1.00 9.42 ATOM491 C GLY 143 5.506 −4.804 23.404 1.00 8.75 ATOM 492 O GLY 143 6.626−5.029 23.862 1.00 8.31 ATOM 493 N LYS 144 5.314 −4.321 22.181 1.00 7.56ATOM 494 CA LYS 144 6.437 −4.054 21.293 1.00 6.96 ATOM 495 CB LYS 1445.988 −4.152 19.833 1.00 7.62 ATOM 496 CG LYS 144 7.102 −3.928 18.8151.00 8.80 ATOM 497 CD LYS 144 8.145 −5.037 18.867 1.00 8.64 ATOM 498 CELYS 144 9.230 −4.827 17.819 1.00 9.66 ATOM 499 NZ LYS 144 10.256 −5.91117.843 1.00 11.27 ATOM 500 C LYS 144 7.067 −2.687 21.527 1.00 6.78 ATOM501 O LYS 144 6.392 −1.657 21.454 1.00 6.18 ATOM 502 N LEU 145 8.365−2.680 21.813 1.00 6.40 ATOM 503 CA LEU 145 9.088 −1.428 22.014 1.006.12 ATOM 504 CB LEU 145 10.437 −1.687 22.689 1.00 5.82 ATOM 505 CG LEU145 10.411 −1.994 24.186 1.00 6.37 ATOM 506 CD1 LEU 145 11.800 −2.42324.650 1.00 7.01 ATOM 507 CD2 LEU 145 9.949 −0.756 24.944 1.00 7.23 ATOM508 C LEU 145 9.323 −0.795 20.646 1.00 6.17 ATOM 509 O LEU 145 9.697−1.483 19.695 1.00 6.13 ATOM 510 N TYR 146 9.095 0.509 20.544 1.00 5.68ATOM 511 CA TYR 146 9.300 1.206 19.282 1.00 6.42 ATOM 512 CB TYR 1468.049 1.078 18.395 1.00 6.42 ATOM 513 CG TYR 146 6.860 1.917 18.824 1.006.55 ATOM 514 CD1 TYR 146 6.665 3.197 18.304 1.00 6.78 ATOM 515 CE1 TYR146 5.585 3.982 18.707 1.00 7.64 ATOM 516 CD2 TYR 146 5.943 1.438 19.7581.00 7.13 ATOM 517 CE2 TYR 146 4.857 2.218 20.169 1.00 7.09 ATOM 518 CZTYR 146 4.687 3.487 19.640 1.00 7.80 ATOM 519 OH TYR 146 3.627 4.26920.044 1.00 7.00 ATOM 520 C TYR 146 9.643 2.672 19.536 1.00 6.69 ATOM521 O TYR 146 9.400 3.201 20.625 1.00 6.62 ATOM 522 N GLY 147 10.2323.320 18.538 1.00 5.95 ATOM 523 CA GLY 147 10.593 4.715 18.692 1.00 5.63ATOM 524 C GLY 147 9.469 5.638 18.268 1.00 5.37 ATOM 525 O GLY 147 8.8865.458 17.194 1.00 5.41 ATOM 526 N SER 148 9.159 6.621 19.108 1.00 5.18ATOM 527 CA SER 148 8.104 7.588 18.810 1.00 5.87 ATOM 528 CB SER 1487.063 7.610 19.934 1.00 6.82 ATOM 529 OG SER 148 5.963 8.438 19.586 1.005.87 ATOM 530 C SER 148 8.744 8.965 18.662 1.00 6.42 ATOM 531 O SER 1489.319 9.496 19.612 1.00 7.70 ATOM 532 N PRO 149 8.652 9.561 17.462 1.007.74 ATOM 533 CD PRO 149 7.983 9.012 16.268 1.00 7.72 ATOM 534 CA PRO149 9.228 10.880 17.182 1.00 8.21 ATOM 535 CB PRO 149 9.112 10.98815.662 1.00 8.68 ATOM 536 CG PRO 149 7.866 10.227 15.368 1.00 9.11 ATOM537 C PRO 149 8.561 12.038 17.924 1.00 8.51 ATOM 538 O PRO 149 9.10113.147 17.965 1.00 8.97 ATOM 539 N PHE 150 7.385 11.782 18.493 1.00 8.22ATOM 540 CA PHE 150 6.674 12.791 19.274 1.00 8.18 ATOM 541 CB PHE 1505.525 13.429 18.466 1.00 9.01 ATOM 542 CG PHE 150 4.593 12.444 17.8201.00 10.81 ATOM 543 CD1 PHE 150 3.496 11.943 18.513 1.00 11.41 ATOM 544CD2 PHE 150 4.798 12.037 16.506 1.00 11.41 ATOM 545 CE1 PHE 150 2.61011.049 17.903 1.00 12.19 ATOM 546 CE2 PHE 150 3.922 11.144 15.886 1.0012.69 ATOM 547 CZ PHE 150 2.824 10.650 16.588 1.00 12.71 ATOM 548 C PHE150 6.173 12.145 20.567 1.00 7.43 ATOM 549 O PHE 150 5.993 10.928 20.6361.00 6.84 ATOM 550 N PHE 151 5.967 12.965 21.593 1.00 6.95 ATOM 551 CAPHE 151 5.550 12.482 22.908 1.00 7.33 ATOM 552 CB PHE 151 5.862 13.56723.945 1.00 8.43 ATOM 553 CG PHE 151 5.952 13.062 25.355 1.00 8.59 ATOM554 CD1 PHE 151 6.480 11.802 25.631 1.00 8.15 ATOM 555 CD2 PHE 151 5.55813.870 26.418 1.00 10.06 ATOM 556 CE1 PHE 151 6.614 11.356 26.943 1.008.82 ATOM 557 CE2 PHE 151 5.690 13.433 27.738 1.00 9.79 ATOM 558 CZ PHE151 6.219 12.174 28.000 1.00 10.29 ATOM 559 C PHE 151 4.086 12.05922.994 1.00 7.03 ATOM 560 O PHE 151 3.192 12.832 22.657 1.00 6.91 ATOM561 N THR 152 3.852 10.827 23.450 1.00 7.20 ATOM 562 CA THR 152 2.49510.286 23.581 1.00 6.32 ATOM 563 CB THR 152 2.152 9.316 22.443 1.00 6.44ATOM 564 OG1 THR 152 2.906 8.110 22.615 1.00 5.93 ATOM 565 CG2 THR 1522.470 9.933 21.090 1.00 7.16 ATOM 566 C THR 152 2.321 9.499 24.872 1.006.73 ATOM 567 O THR 152 3.273 9.306 25.629 1.00 6.29 ATOM 568 N ASP 1531.102 9.013 25.097 1.00 7.13 ATOM 569 CA ASP 153 0.801 8.240 26.292 1.007.68 ATOM 570 CB ASP 153 −0.720 8.176 26.521 1.00 8.99 ATOM 571 CG ASP153 −1.477 7.518 25.379 1.00 9.77 ATOM 572 OD1 ASP 153 −0.949 7.43724.252 1.00 9.30 ATOM 573 OD2 ASP 153 −2.631 7.095 25.614 1.00 10.99ATOM 574 C ASP 153 1.424 6.837 26.297 1.00 7.99 ATOM 575 O ASP 153 1.3046.110 27.282 1.00 8.34 ATOM 576 N GLU 154 2.092 6.458 25.208 1.00 7.55ATOM 577 CA GLU 154 2.763 5.157 25.157 1.00 6.93 ATOM 578 CB GLU 1542.537 4.456 23.806 1.00 7.67 ATOM 579 CG GLU 154 1.090 4.029 23.552 1.007.08 ATOM 580 CD GLU 154 0.944 3.048 22.395 1.00 7.82 ATOM 581 OE1 GLU154 1.765 3.091 21.456 1.00 6.93 ATOM 582 OE2 GLU 154 −0.010 2.23722.417 1.00 7.76 ATOM 583 C GLU 154 4.265 5.353 25.395 1.00 6.91 ATOM584 O GLU 154 5.048 4.409 25.276 1.00 6.34 ATOM 585 N CYS 155 4.6536.579 25.746 1.00 6.29 ATOM 586 CA CYS 155 6.055 6.918 26.003 1.00 6.66ATOM 587 CB CYS 155 6.426 8.221 25.292 1.00 6.30 ATOM 588 SG CYS 1556.252 8.217 23.510 1.00 7.00 ATOM 589 C CYS 155 6.352 7.099 27.492 1.007.01 ATOM 590 O CYS 155 7.441 7.538 27.863 1.00 8.21 ATOM 591 N THR 1565.386 6.775 28.340 1.00 6.69 ATOM 592 CA THR 156 5.560 6.931 29.780 1.005.87 ATOM 593 CB THR 156 4.348 7.646 30.391 1.00 5.85 ATOM 594 OG1 THR156 3.146 7.045 29.903 1.00 6.18 ATOM 595 CG2 THR 156 4.360 9.118 30.0101.00 6.61 ATOM 596 C THR 156 5.763 5.589 30.467 1.00 5.83 ATOM 597 O THR156 5.073 4.615 30.167 1.00 5.54 ATOM 598 N PHE 157 6.710 5.553 31.4011.00 5.70 ATOM 599 CA PHE 157 7.043 4.327 32.113 1.00 5.65 ATOM 600 CBPHE 157 8.341 3.739 31.545 1.00 6.27 ATOM 601 CG PHE 157 8.240 3.31430.111 1.00 5.92 ATOM 602 CD1 PHE 157 7.775 2.046 29.779 1.00 6.03 ATOM603 CD2 PHE 157 8.582 4.192 29.089 1.00 5.50 ATOM 604 CE1 PHE 157 7.6501.658 28.445 1.00 6.23 ATOM 605 CE2 PHE 157 8.459 3.814 27.758 1.00 5.35ATOM 606 CZ PHE 157 7.992 2.543 27.437 1.00 5.57 ATOM 607 C PHE 1577.244 4.545 33.604 1.00 6.10 ATOM 608 O PHE 157 7.811 5.553 34.021 1.005.75 ATOM 609 N LYS 158 6.784 3.590 34.403 1.00 6.36 ATOM 610 CA LYS 1586.976 3.661 35.843 1.00 6.76 ATOM 611 CB LYS 158 5.964 2.772 36.567 1.007.54 ATOM 612 CG LYS 158 4.531 3.239 36.431 1.00 8.41 ATOM 613 CD LYS158 3.599 2.436 37.322 1.00 10.05 ATOM 614 CE LYS 158 2.186 2.986 37.2471.00 11.40 ATOM 615 NZ LYS 158 1.286 2.332 38.237 1.00 12.48 ATOM 616 CLYS 158 8.389 3.153 36.111 1.00 6.88 ATOM 617 O LYS 158 8.743 2.04835.698 1.00 7.51 ATOM 618 N GLU 159 9.195 3.966 36.785 1.00 6.50 ATOM619 CA GLU 159 10.568 3.598 37.117 1.00 7.08 ATOM 620 CB GLU 159 11.4464.847 37.124 1.00 8.45 ATOM 621 CG GLU 159 12.930 4.578 37.312 1.0010.72 ATOM 622 CD GLU 159 13.754 5.830 37.101 1.00 12.99 ATOM 623 OE1GLU 159 13.516 6.820 37.823 1.00 14.61 ATOM 624 OE2 GLU 159 14.632 5.82936.212 1.00 14.45 ATOM 625 C GLU 159 10.535 2.954 38.498 1.00 7.00 ATOM626 O GLU 159 10.389 3.639 39.515 1.00 6.45 ATOM 627 N ILE 160 10.6791.632 38.521 1.00 6.44 ATOM 628 CA ILE 160 10.602 0.850 39.749 1.00 6.73ATOM 629 CB ILE 160 9.649 −0.348 39.534 1.00 7.25 ATOM 630 CG2 ILE 1609.493 −1.142 40.827 1.00 8.05 ATOM 631 CG1 ILE 160 8.292 0.169 39.0471.00 7.90 ATOM 632 CD1 ILE 160 7.370 −0.906 38.490 1.00 8.51 ATOM 633 CILE 160 11.946 0.336 40.245 1.00 7.21 ATOM 634 O ILE 160 12.623 −0.42639.553 1.00 6.33 ATOM 635 N LEU 161 12.307 0.742 41.461 1.00 6.48 ATOM636 CA LEU 161 13.570 0.337 42.065 1.00 6.42 ATOM 637 CB LEU 161 13.8201.115 43.362 1.00 6.84 ATOM 638 CG LEU 161 15.159 0.811 44.047 1.00 6.63ATOM 639 CD1 LEU 161 16.301 1.238 43.135 1.00 7.13 ATOM 640 CD2 LEU 16115.247 1.538 45.382 1.00 7.21 ATOM 641 C LEU 161 13.618 −1.154 42.3631.00 7.01 ATOM 642 O LEU 161 12.632 −1.745 42.816 1.00 5.88 ATOM 643 NLEU 162 14.785 −1.741 42.110 1.00 6.67 ATOM 644 CA LEU 162 15.044 −3.16042.333 1.00 7.48 ATOM 645 CB LEU 162 15.362 −3.843 41.000 1.00 7.02 ATOM646 CG LEU 162 14.313 −3.734 39.893 1.00 7.49 ATOM 647 CD1 LEU 16214.921 −4.162 38.563 1.00 7.87 ATOM 648 CD2 LEU 162 13.112 −4.591 40.2541.00 8.37 ATOM 649 C LEU 162 16.264 −3.270 43.243 1.00 7.49 ATOM 650 OLEU 162 16.890 −2.264 43.570 1.00 7.10 ATOM 651 N PRO 163 16.610 −4.49343.677 1.00 8.02 ATOM 652 CD PRO 163 15.853 −5.756 43.606 1.00 8.20 ATOM653 CA PRO 163 17.785 −4.635 44.542 1.00 8.81 ATOM 654 CB PRO 163 17.822−6.135 44.827 1.00 9.08 ATOM 655 CG PRO 163 16.371 −6.514 44.808 1.008.96 ATOM 656 C PRO 163 19.032 −4.175 43.784 1.00 9.09 ATOM 657 O PRO163 19.061 −4.205 42.554 1.00 8.86 ATOM 658 N ASN 164 20.049 −3.74344.522 1.00 9.58 ATOM 659 CA ASN 164 21.314 −3.303 43.936 1.00 10.43ATOM 660 CB ASN 164 22.031 −4.499 43.304 1.00 11.05 ATOM 661 CG ASN 16422.486 −5.518 44.338 1.00 12.27 ATOM 662 OD1 ASN 164 22.849 −6.64743.999 1.00 13.69 ATOM 663 ND2 ASN 164 22.478 −5.119 45.605 1.00 10.90ATOM 664 C ASN 164 21.246 −2.144 42.935 1.00 10.78 ATOM 665 O ASN 16422.006 −2.098 41.965 1.00 11.04 ATOM 666 N ASN 165 20.332 −1.215 43.1931.00 10.80 ATOM 667 CA ASN 165 20.148 −0.009 42.390 1.00 11.74 ATOM 668CB ASN 165 21.426 0.836 42.423 1.00 14.07 ATOM 669 CG ASN 165 21.1422.328 42.341 1.00 17.00 ATOM 670 OD1 ASN 165 20.431 2.884 43.187 1.0017.76 ATOM 671 ND2 ASN 165 21.691 2.983 41.322 1.00 18.46 ATOM 672 C ASN165 19.686 −0.178 40.944 1.00 10.73 ATOM 673 O ASN 165 19.738 0.76940.162 1.00 11.09 ATOM 674 N TYR 166 19.245 −1.375 40.575 1.00 9.88 ATOM675 CA TYR 166 18.737 −1.586 39.221 1.00 9.07 ATOM 676 CB TYR 166 18.721−3.073 38.862 1.00 9.58 ATOM 677 CG TYR 166 20.043 −3.623 38.390 1.0011.15 ATOM 678 CD1 TYR 166 20.312 −3.767 37.028 1.00 11.97 ATOM 679 CE1TYR 166 21.533 −4.271 36.582 1.00 13.55 ATOM 680 CD2 TYR 166 21.032−3.991 39.300 1.00 13.15 ATOM 681 CE2 TYR 166 22.257 −4.493 38.866 1.0014.56 ATOM 682 CZ TYR 166 22.499 −4.630 37.506 1.00 14.88 ATOM 683 OHTYR 166 23.707 −5.134 37.068 1.00 16.64 ATOM 684 C TYR 166 17.302 −1.07639.218 1.00 8.86 ATOM 685 O TYR 166 16.690 −0.947 40.277 1.00 9.39 ATOM686 N ASN 167 16.772 −0.790 38.032 1.00 7.93 ATOM 687 CA ASN 167 15.396−0.332 37.895 1.00 8.47 ATOM 688 CB ASN 167 15.332 1.143 37.480 1.009.78 ATOM 689 CG ASN 167 15.806 2.084 38.561 1.00 11.84 ATOM 690 OD1 ASN167 15.313 2.055 39.687 1.00 13.23 ATOM 691 ND2 ASN 167 16.760 2.93938.218 1.00 13.91 ATOM 692 C ASN 167 14.688 −1.136 36.818 1.00 7.74 ATOM693 O ASN 167 15.313 −1.607 35.863 1.00 8.53 ATOM 694 N ALA 168 13.381−1.302 36.989 1.00 7.42 ATOM 695 CA ALA 168 12.559 −1.972 35.995 1.005.36 ATOM 696 CB ALA 168 11.664 −3.014 36.640 1.00 5.62 ATOM 697 C ALA168 11.720 −0.827 35.451 1.00 5.33 ATOM 698 O ALA 168 11.470 0.15536.158 1.00 5.24 ATOM 699 N TYR 169 11.299 −0.939 34.199 1.00 4.83 ATOM700 CA TYR 169 10.485 0.100 33.583 1.00 5.05 ATOM 701 CB TYR 169 11.2700.770 32.453 1.00 5.13 ATOM 702 CG TYR 169 12.432 1.583 32.983 1.00 5.77ATOM 703 CD1 TYR 169 12.254 2.900 33.403 1.00 6.00 ATOM 704 CE1 TYR 16913.295 3.617 33.994 1.00 6.57 ATOM 705 CD2 TYR 169 13.686 1.001 33.1571.00 5.97 ATOM 706 CE2 TYR 169 14.733 1.706 33.745 1.00 6.85 ATOM 707 CZTYR 169 14.533 3.009 34.164 1.00 7.84 ATOM 708 OH TYR 169 15.568 3.69034.774 1.00 8.35 ATOM 709 C TYR 169 9.200 −0.524 33.071 1.00 5.40 ATOM710 O TYR 169 9.207 −1.297 32.114 1.00 5.61 ATOM 711 N GLU 170 8.095−0.185 33.723 1.00 5.44 ATOM 712 CA GLU 170 6.794 −0.733 33.362 1.006.45 ATOM 713 CB GLU 170 6.052 −1.154 34.635 1.00 7.61 ATOM 714 CG GLU170 4.608 −1.600 34.426 1.00 7.81 ATOM 715 CD GLU 170 3.905 −1.89735.740 1.00 8.83 ATOM 716 OE1 GLU 170 4.305 −1.316 36.772 1.00 9.65 ATOM717 OE2 GLU 170 2.947 −2.698 35.742 1.00 9.00 ATOM 718 C GLU 170 5.9470.255 32.574 1.00 6.80 ATOM 719 O GLU 170 5.883 1.441 32.906 1.00 6.00ATOM 720 N SER 171 5.304 −0.235 31.521 1.00 6.67 ATOM 721 CA SER 1714.445 0.615 30.709 1.00 7.04 ATOM 722 CB SER 171 3.755 −0.201 29.6151.00 6.44 ATOM 723 CG SER 171 2.748 0.577 28.982 1.00 8.08 ATOM 724 CSER 171 3.380 1.241 31.593 1.00 7.11 ATOM 725 O SER 171 2.683 0.54132.330 1.00 7.84 ATOM 726 N TYR 172 3.247 2.560 31.516 1.00 7.12 ATOM727 CA TYR 172 2.246 3.243 32.314 1.00 8.72 ATOM 728 CB TYR 172 2.5074.748 32.327 1.00 9.00 ATOM 729 CG TYR 172 1.543 5.499 33.207 1.00 11.05ATOM 730 CD1 TYR 172 1.765 5.613 34.578 1.00 11.87 ATOM 731 CE1 TYR 1720.849 6.260 35.403 1.00 13.42 ATOM 732 CD2 TYR 172 0.378 6.050 32.6781.00 12.30 ATOM 733 CE2 TYR 172 −0.543 6.695 33.492 1.00 14.29 ATOM 734CZ TYR 172 −0.303 6.797 34.850 1.00 14.57 ATOM 735 OH TYR 172 −1.2207.438 35.652 1.00 17.42 ATOM 736 C TYR 172 0.855 2.969 31.738 1.00 8.33ATOM 737 O TYR 172 −0.103 2.751 32.477 1.00 8.14 ATOM 738 N LYS 1730.748 2.975 30.414 1.00 8.55 ATOM 739 CA LYS 173 −0.536 2.734 29.7641.00 8.99 ATOM 740 CB LYS 173 −0.496 3.209 28.306 1.00 9.71 ATOM 741 CGLYS 173 −1.848 3.137 27.605 1.00 10.52 ATOM 742 CD LYS 173 −1.861 3.93226.300 1.00 10.91 ATOM 743 CE LYS 173 −3.273 4.008 25.727 1.00 11.42ATOM 744 NZ LYS 173 −3.348 4.800 24.469 1.00 11.20 ATOM 745 C LYS 173−0.977 1.273 29.808 1.00 8.99 ATOM 746 O LYS 173 −2.177 0.985 29.8491.00 7.88 ATOM 747 N TYR 174 −0.012 0.355 29.797 1.00 8.58 ATOM 748 CATYR 174 −0.302 −1.080 29.818 1.00 9.01 ATOM 749 CB TYR 174 0.170 −1.72428.510 1.00 8.57 ATOM 750 CG TYR 174 −0.329 −0.998 27.285 1.00 8.58 ATOM751 CD1 TYR 174 −1.691 −0.947 26.988 1.00 7.77 ATOM 752 CE1 TYR 174−2.162 −0.224 25.894 1.00 7.99 ATOM 753 CD2 TYR 174 0.555 −0.314 26.4501.00 7.31 ATOM 754 CE2 TYR 174 0.095 0.411 25.354 1.00 7.84 ATOM 755 CZTYR 174 −1.263 0.454 25.083 1.00 7.62 ATOM 756 OH TYR 174 −1.721 1.18524.014 1.00 7.92 ATOM 757 C TYR 174 0.374 −1.771 31.002 1.00 9.15 ATOM758 O TYR 174 1.466 −2.332 30.873 1.00 8.77 ATOM 759 N PRO 175 −0.277−1.742 32.176 1.00 9.16 ATOM 760 CD PRO 175 −1.589 −1.129 32.440 1.009.90 ATOM 761 CA PRO 175 0.256 −2.361 33.391 1.00 9.10 ATOM 762 CB PRO175 −0.905 −2.227 34.375 1.00 10.40 ATOM 763 CG PRO 175 −1.566 −0.96633.941 1.00 10.31 ATOM 764 C PRO 175 0.663 −3.813 33.180 1.00 9.23 ATOM765 O PRO 175 0.000 −4.556 32.455 1.00 9.54 ATOM 766 N GLY 176 1.765−4.207 33.806 1.00 9.48 ATOM 767 CA GLY 176 2.226 −5.577 33.690 1.009.58 ATOM 768 C GLY 176 3.192 −5.838 32.554 1.00 9.81 ATOM 769 O GLY 1763.677 −6.958 32.409 1.00 10.30 ATOM 770 N MET 177 3.463 −4.823 31.7391.00 8.82 ATOM 771 CA MET 177 4.401 −4.975 30.629 1.00 8.52 ATOM 772 CBMET 177 3.760 −4.515 29.320 1.00 10.08 ATOM 773 CG MET 177 2.649 −5.43928.859 1.00 11.46 ATOM 774 SD MET 177 2.093 −5.117 27.179 1.00 12.85ATOM 775 CE MET 177 0.393 −5.651 27.292 1.00 13.23 ATOM 776 C MET 1775.660 −4.172 30.930 1.00 7.98 ATOM 777 O MET 177 5.579 −3.010 31.3261.00 7.40 ATOM 778 N PHE 178 6.820 −4.797 30.739 1.00 7.68 ATOM 779 CAPHE 178 8.094 −4.160 31.049 1.00 7.09 ATOM 780 CB PHE 178 8.743 −4.86432.250 1.00 8.40 ATOM 781 CG PHE 178 7.874 −4.908 33.477 1.00 9.38 ATOM782 CD1 PHE 178 6.767 −5.754 33.536 1.00 9.55 ATOM 783 CD2 PHE 178 8.150−4.090 34.569 1.00 9.47 ATOM 784 CE1 PHE 178 5.947 −5.783 34.664 1.009.89 ATOM 785 CE2 PHE 178 7.336 −4.112 35.699 1.00 10.43 ATOM 786 CZ PHE178 6.232 −4.960 35.746 1.00 9.58 ATOM 787 C PHE 178 9.107 −4.119 29.9141.00 7.00 ATOM 788 O PHE 178 9.128 −4.993 29.044 1.00 6.97 ATOM 789 NILE 179 9.955 −3.094 29.943 1.00 6.62 ATOM 790 CA ILE 179 11.010 −2.92528.949 1.00 6.43 ATOM 791 CB ILE 179 11.736 −1.569 29.124 1.00 6.72 ATOM792 CG2 ILE 179 12.947 −1.496 28.202 1.00 7.70 ATOM 793 CG1 ILE 17910.767 −0.418 28.847 1.00 6.16 ATOM 794 CD1 ILE 179 11.411 0.966 28.9331.00 6.64 ATOM 795 C ILE 179 12.017 −4.038 29.195 1.00 6.58 ATOM 796 OILE 179 12.330 −4.344 30.342 1.00 6.50 ATOM 797 N ALA 180 12.531 −4.63928.129 1.00 6.56 ATOM 798 CA ALA 180 13.496 −5.712 28.302 1.00 6.46 ATOM799 CB ALA 180 12.770 −7.005 28.677 1.00 6.63 ATOM 800 C ALA 180 14.369−5.953 27.082 1.00 6.85 ATOM 801 O ALA 180 13.950 −5.731 25.945 1.006.24 ATOM 802 N LEU 181 15.593 −6.405 27.344 1.00 6.67 ATOM 803 CA LEU181 16.561 −6.745 26.305 1.00 7.47 ATOM 804 CB LEU 181 17.795 −5.83926.379 1.00 7.48 ATOM 805 CG LEU 181 17.654 −4.375 25.964 1.00 7.58 ATOM806 CD1 LEU 181 18.985 −3.680 26.176 1.00 7.44 ATOM 807 CD2 LEU 18117.230 −4.274 24.503 1.00 8.28 ATOM 808 C LEU 181 16.987 −8.186 26.5711.00 8.19 ATOM 809 O LEU 181 17.144 −8.591 27.725 1.00 8.34 ATOM 810 NALA 182 17.176 −8.956 25.508 1.00 9.76 ATOM 811 CA ALA 182 17.583−10.346 25.648 1.00 9.78 ATOM 812 CB ALA 182 16.934 −11.180 24.570 1.0013.35 ATOM 813 C ALA 182 19.095 −10.461 25.558 1.00 10.87 ATOM 814 O ALA182 19.778 −9.487 25.243 1.00 8.94 ATOM 815 N LYS 183 19.616 −11.65725.825 1.00 11.33 ATOM 816 CA LYS 183 21.054 −11.872 25.779 1.00 12.77ATOM 817 CB LYS 183 21.402 −13.275 26.299 1.00 13.33 ATOM 818 CG LYS 18320.846 −14.427 25.487 1.00 14.58 ATOM 819 CD LYS 183 21.231 −15.76026.124 1.00 15.82 ATOM 820 CE LYS 183 20.900 −16.937 25.222 1.00 17.00ATOM 821 NZ LYS 183 19.446 −17.024 24.917 1.00 18.11 ATOM 822 C LYS 18321.645 −11.650 24.389 1.00 13.15 ATOM 823 O LYS 183 22.855 −11.45824.254 1.00 14.24 ATOM 824 N ASN 184 20.804 −11.668 23.356 1.00 13.74ATOM 825 CA ASN 184 21.291 −11.440 21.998 1.00 14.34 ATOM 826 CB ASN 18420.399 −12.141 20.960 1.00 15.53 ATOM 827 CG ASN 184 18.962 −11.65720.985 1.00 16.25 ATOM 828 OD1 ASN 184 18.628 −10.692 21.668 1.00 16.76ATOM 829 ND2 ASN 184 18.102 −12.329 20.229 1.00 17.81 ATOM 830 C ASN 18421.369 −9.943 21.701 1.00 14.45 ATOM 831 O ASN 184 21.711 −9.535 20.5901.00 14.60 ATOM 832 N GLY 185 21.051 −9.132 22.708 1.00 13.68 ATOM 833CA GLY 185 21.109 −7.688 22.557 1.00 13.05 ATOM 834 C GLY 185 19.904−7.049 21.894 1.00 12.79 ATOM 835 O GLY 185 19.911 −5.848 21.631 1.0013.80 ATOM 836 N ALA 186 18.873 −7.841 21.619 1.00 10.99 ATOM 837 CA ALA186 17.670 −7.318 20.985 1.00 10.27 ATOM 838 CB ALA 186 17.246 −8.22519.828 1.00 10.89 ATOM 839 C ALA 186 16.545 −7.197 22.004 1.00 8.96 ATOM840 O ALA 186 16.578 −7.820 23.063 1.00 8.77 ATOM 841 N THR 187 15.545−6.385 21.687 1.00 8.86 ATOM 842 CA THR 187 14.427 −6.203 22.600 1.007.58 ATOM 843 CB THR 187 13.518 −5.051 22.146 1.00 7.14 ATOM 844 OG1 THR187 13.033 −5.325 20.825 1.00 7.19 ATOM 845 CG2 THR 187 14.281 −3.73422.146 1.00 6.53 ATOM 846 C THR 187 13.562 −7.452 22.700 1.00 8.41 ATOM847 O THR 187 13.527 −8.279 21.788 1.00 8.64 ATOM 848 N LYS 188 12.876−7.585 23.829 1.00 8.14 ATOM 849 CA LYS 188 11.953 −8.684 24.056 1.009.20 ATOM 850 CB LYS 188 12.271 −9.412 25.363 1.00 10.91 ATOM 851 CG LYS188 13.456 −10.356 25.287 1.00 12.37 ATOM 852 CD LYS 188 13.577 −11.14026.583 1.00 14.48 ATOM 853 CE LYS 188 14.470 −12.345 26.416 1.00 16.86ATOM 854 NZ LYS 188 13.932 −13.324 25.422 1.00 15.93 ATOM 855 C LYS 18810.588 −8.020 24.167 1.00 9.15 ATOM 856 O LYS 188 10.506 −6.823 24.4431.00 8.81 ATOM 857 N LYS 189 9.523 −8.783 23.944 1.00 8.51 ATOM 858 CALYS 189 8.170 −8.242 24.040 1.00 9.55 ATOM 859 CB LYS 189 7.164 −9.23723.452 1.00 11.24 ATOM 860 CG LYS 189 7.433 −9.583 21.991 1.00 13.77ATOM 861 CD LYS 189 7.115 −8.412 21.070 1.00 15.40 ATOM 862 CE LYS 1895.666 −8.448 20.601 1.00 18.07 ATOM 863 NZ LYS 189 4.706 −8.523 21.7291.00 20.82 ATOM 864 C LYS 189 7.842 −7.965 25.506 1.00 8.74 ATOM 865 OLYS 189 8.100 −8.799 26.372 1.00 8.90 ATOM 866 N GLY 190 7.273 −6.79325.776 1.00 7.88 ATOM 867 CA GLY 190 6.940 −6.421 27.140 1.00 8.10 ATOM868 C GLY 190 5.938 −7.314 27.851 1.00 8.54 ATOM 869 O GLY 190 5.894−7.339 29.086 1.00 7.39 ATOM 870 N ASN 191 5.130 −8.045 27.090 1.00 8.98ATOM 871 CA ASN 191 4.135 −8.926 27.689 1.00 10.89 ATOM 872 CB ASN 1912.812 −8.829 26.922 1.00 12.28 ATOM 873 CG ASN 191 2.937 −9.274 25.4831.00 13.78 ATOM 874 OD1 ASN 191 4.035 −9.334 24.931 1.00 13.27 ATOM 875ND2 ASN 191 1.803 −9.575 24.858 1.00 15.88 ATOM 876 C ASN 191 4.607−10.377 27.756 1.00 11.62 ATOM 877 O ASN 191 3.802 −11.295 27.916 1.0010.96 ATOM 878 N ARG 192 5.917 −10.574 27.639 1.00 12.11 ATOM 879 CA ARG192 6.503 −11.910 27.715 1.00 13.33 ATOM 880 CB ARG 192 7.076 −12.32426.356 1.00 14.44 ATOM 881 CG ARG 192 6.026 −12.464 25.263 1.00 16.19ATOM 882 CD ARG 192 6.663 −12.877 23.948 1.00 19.13 ATOM 883 NE ARG 1927.220 −14.224 24.013 1.00 22.54 ATOM 884 CZ ARG 192 6.499 −15.337 23.9051.00 24.09 ATOM 885 NH1 ARG 192 5.188 −15.266 23.725 1.00 24.60 ATOM 886NH2 ARG 192 7.091 −16.521 23.977 1.00 25.22 ATOM 887 C ARG 192 7.597−11.957 28.780 1.00 13.36 ATOM 888 O ARG 192 8.358 −12.919 28.861 1.0013.48 ATOM 889 N VAL 193 7.663 −10.908 29.596 1.00 13.03 ATOM 890 CA VAL193 8.654 −10.812 30.665 1.00 12.85 ATOM 891 CB VAL 193 9.800 −9.84130.284 1.00 11.28 ATOM 892 CG1 VAL 193 10.543 −10.360 29.064 1.00 11.22ATOM 893 CG2 VAL 193 9.235 −8.457 30.003 1.00 11.65 ATOM 894 C VAL 1938.010 −10.305 31.957 1.00 13.63 ATOM 895 O VAL 193 6.958 −9.660 31.9271.00 14.01 ATOM 896 N SER 194 8.647 −10.605 33.086 1.00 14.34 ATOM 897CA SER 194 8.166 −10.172 34.398 1.00 15.34 ATOM 898 CB SER 194 7.728−11.372 35.238 1.00 16.67 ATOM 899 OG SER 194 8.833 −12.193 35.565 1.0019.38 ATOM 900 C SER 194 9.315 −9.453 35.095 1.00 14.47 ATOM 901 O SER194 10.480 −9.720 34.810 1.00 13.57 ATOM 902 N PRO 195 9.003 −8.54536.034 1.00 14.17 ATOM 903 CD PRO 195 7.669 −8.211 36.562 1.00 14.38ATOM 904 CA PRO 195 10.048 −7.804 36.745 1.00 13.64 ATOM 905 CB PRO 1959.252 −6.826 37.607 1.00 14.45 ATOM 906 CG PRO 195 8.002 −7.588 37.9001.00 14.65 ATOM 907 C PRO 195 11.033 −8.638 37.557 1.00 13.10 ATOM 908 OPRO 195 12.046 −8.118 38.007 1.00 12.82 ATOM 909 N THR 196 10.746 −9.92137.753 1.00 12.72 ATOM 910 CA THR 196 11.666 −10.771 38.509 1.00 12.30ATOM 911 CB THR 196 10.950 −11.988 39.121 1.00 12.60 ATOM 912 OG1 THR196 10.332 −12.753 38.079 1.00 12.75 ATOM 913 CG2 THR 196 9.898 −11.54040.121 1.00 13.99 ATOM 914 C THR 196 12.785 −11.283 37.611 1.00 12.27ATOM 915 O THR 196 13.798 −11.786 38.094 1.00 12.60 ATOM 916 N MET 19712.593 −11.138 36.303 1.00 11.81 ATOM 917 CA MET 197 13.561 −11.59635.309 1.00 11.06 ATOM 918 CB MET 197 12.832 −11.920 34.005 1.00 11.47ATOM 919 CG MET 197 11.814 −13.042 34.142 1.00 13.03 ATOM 920 SD MET 19710.747 −13.178 32.697 1.00 13.94 ATOM 921 CE MET 197 11.878 −13.88431.521 1.00 14.56 ATOM 922 C MET 197 14.662 −10.574 35.050 1.00 10.53ATOM 923 O MET 197 14.399 −9.379 34.943 1.00 9.30 ATOM 924 N ALA 19815.896 −11.060 34.942 1.00 9.44 ATOM 925 CA ALA 198 17.051 −10.20534.708 1.00 8.58 ATOM 926 CB ALA 198 18.327 −11.049 34.690 1.00 9.44ATOM 927 C ALA 198 16.958 −9.378 33.429 1.00 8.75 ATOM 928 O ALA 19817.544 −8.296 33.349 1.00 8.37 ATOM 929 N VAL 199 16.234 −9.877 32.4311.00 7.63 ATOM 930 CA VAL 199 16.097 −9.152 31.170 1.00 6.86 ATOM 931 CBVAL 199 15.388 −10.017 30.092 1.00 7.52 ATOM 932 CG1 VAL 199 16.272−11.193 29.714 1.00 8.00 ATOM 933 CG2 VAL 199 14.043 −10.511 30.608 1.007.54 ATOM 934 C VAL 199 15.353 −7.819 31.325 1.00 6.93 ATOM 935 O VAL199 15.382 −6.981 30.421 1.00 5.85 ATOM 936 N THR 200 14.704 −7.61532.470 1.00 6.77 ATOM 937 CA THR 200 13.972 −6.369 32.721 1.00 7.57 ATOM938 CB THR 200 12.578 −6.631 33.340 1.00 8.45 ATOM 939 OG1 THR 20012.736 −7.158 34.664 1.00 8.56 ATOM 940 CG2 THR 200 11.790 −7.622 32.5001.00 8.63 ATOM 941 C THR 200 14.725 −5.457 33.692 1.00 7.78 ATOM 942 OTHR 200 14.247 −4.370 34.028 1.00 7.71 ATOM 943 N HIS 201 15.899 −5.89634.137 1.00 7.34 ATOM 944 CA HIS 201 16.695 −5.125 35.095 1.00 7.59 ATOM945 CB HIS 201 17.452 −6.073 36.028 1.00 8.17 ATOM 946 CG HIS 201 16.568−6.922 36.887 1.00 9.32 ATOM 947 CD2 HIS 201 16.861 −7.736 37.930 1.009.55 ATOM 948 ND1 HIS 201 15.205 −7.007 36.709 1.00 10.97 ATOM 949 CE1HIS 201 14.695 −7.834 37.604 1.00 9.67 ATOM 950 NE2 HIS 201 15.681−8.290 38.358 1.00 11.80 ATOM 951 C HIS 201 17.704 −4.199 34.426 1.007.38 ATOM 952 O HIS 201 18.618 −4.663 33.742 1.00 7.78 ATOM 953 N PHE202 17.553 −2.894 34.640 1.00 7.48 ATOM 954 CA PHE 202 18.466 −1.92234.048 1.00 7.69 ATOM 955 CB PHE 202 17.716 −0.988 33.091 1.00 7.39 ATOM956 CG PHE 202 17.187 −1.673 31.866 1.00 7.17 ATOM 957 CD1 PHE 20215.966 −2.334 31.894 1.00 7.73 ATOM 958 CD2 PHE 202 17.922 −1.674 30.6861.00 6.55 ATOM 959 CE1 PHE 202 15.480 −2.988 30.765 1.00 6.46 ATOM 960CE2 PHE 202 17.447 −2.327 29.545 1.00 6.72 ATOM 961 CZ PHE 202 16.220−2.986 29.587 1.00 6.52 ATOM 962 C PHE 202 19.188 −1.080 35.092 1.008.95 ATOM 963 O PHE 202 18.561 −0.494 35.980 1.00 9.11 ATOM 964 N LEU203 20.511 −1.019 34.972 1.00 9.21 ATOM 965 CA LEU 203 21.334 −0.23735.890 1.00 10.82 ATOM 966 CB LEU 203 22.627 −0.989 36.224 1.00 10.86ATOM 967 CG LEU 203 23.571 −0.292 37.213 1.00 11.00 ATOM 968 CD1 LEU 20322.887 −0.188 38.568 1.00 12.23 ATOM 969 CD2 LEU 203 24.871 −1.06937.341 1.00 11.91 ATOM 970 C LEU 203 21.689 1.103 35.256 1.00 12.02 ATOM971 O LEU 203 22.303 1.149 34.191 1.00 11.12 ATOM 972 N PRO 204 21.2882.214 35.892 1.00 13.88 ATOM 973 CD PRO 204 20.358 2.318 37.029 1.0014.72 ATOM 974 CA PRO 204 21.596 3.540 35.351 1.00 16.16 ATOM 975 CB PRO204 20.832 4.478 36.280 1.00 16.31 ATOM 976 CG PRO 204 19.679 3.63236.748 1.00 15.39 ATOM 977 C PRO 204 23.101 3.786 35.409 1.00 18.73 ATOM978 O PRO 204 23.716 3.625 36.466 1.00 18.38 ATOM 979 N ARG 205 23.6884.165 34.278 1.00 21.09 ATOM 980 CA ARG 205 25.123 4.432 34.213 1.0024.19 ATOM 981 CB ARG 205 25.867 3.219 33.650 1.00 25.55 ATOM 982 CG ARG205 25.752 1.972 34.509 1.00 28.06 ATOM 983 CD ARG 205 27.109 1.32934.774 1.00 30.17 ATOM 984 NE ARG 205 28.040 2.244 35.432 1.00 31.85ATOM 985 CZ ARG 205 28.888 3.045 34.792 1.00 33.10 ATOM 986 NH1 ARG 20528.933 3.050 33.464 1.00 33.73 ATOM 987 NH2 ARG 205 29.690 3.849 35.4801.00 33.12 ATOM 988 C ARG 205 25.442 5.659 33.366 1.00 25.09 ATOM 989 OARG 205 24.577 6.174 32.659 1.00 24.94 ATOM 990 N ALA 206 26.690 6.11733.459 1.00 26.79 ATOM 991 CA ALA 206 27.188 7.281 32.722 1.00 28.01ATOM 992 CB ALA 206 28.274 6.843 31.745 1.00 28.80 ATOM 993 C ALA 20626.107 8.065 31.983 1.00 28.96 ATOM 994 O ALA 206 26.376 8.463 30.8291.00 29.66 ATOM 995 O1 HOH 1000 12.829 −2.036 17.116 1.00 7.18 ATOM 996O1 HOH 1001 20.982 8.993 22.203 1.00 7.57 ATOM 997 O1 HOH 1002 12.76812.229 28.379 1.00 10.08 ATOM 998 O1 HOH 1003 21.297 3.228 27.387 1.006.83 ATOM 999 O1 HOH 1004 2.864 4.158 28.500 1.00 7.52 ATOM 1000 O1 HOH1005 10.142 −1.516 16.908 1.00 7.61 ATOM 1001 O1 HOH 1006 11.860 −3.32719.508 1.00 5.26 ATOM 1002 O1 HOH 1007 9.727 7.593 26.490 1.00 7.03 ATOM1003 O1 HOH 1008 10.236 −4.757 26.340 1.00 7.00 ATOM 1004 O1 HOH 100910.856 2.908 42.825 1.00 6.81 ATOM 1005 O1 HOH 1010 6.747 4.115 39.6951.00 8.95 ATOM 1006 O1 HOH 1011 12.036 −3.230 32.828 1.00 8.27 ATOM 1007O1 HOH 1012 4.734 16.208 33.512 1.00 12.05 ATOM 1008 O1 HOH 1013 10.0930.852 15.744 1.00 8.23 ATOM 1009 O1 HOH 1014 0.631 2.544 19.025 1.0010.23 ATOM 1010 O1 HOH 1015 18.399 −0.846 45.247 1.00 8.81 ATOM 1011 O1HOH 1016 3.640 7.004 20.379 1.00 5.67 ATOM 1012 O1 HOH 1017 19.978−6.895 33.952 1.00 10.33 ATOM 1013 O1 HOH 1018 3.469 1.860 26.778 1.0010.74 ATOM 1014 O1 HOH 1019 19.254 1.746 45.513 1.00 11.57 ATOM 1015 O1HOH 1020 9.787 −5.206 22.223 1.00 9.48 ATOM 1016 O1 HOH 1021 9.165 4.10114.820 1.00 9.74 ATOM 1017 O1 HOH 1022 19.029 7.930 20.394 1.00 7.35ATOM 1018 O1 HOH 1023 11.450 −7.648 40.765 1.00 14.67 ATOM 1019 O1 HOH1024 10.751 −7.011 20.455 1.00 9.99 ATOM 1020 O1 HOH 1025 −0.784 0.04020.721 1.00 11.72 ATOM 1021 O1 HOH 1026 −2.374 3.652 22.178 1.00 16.42ATOM 1022 O1 HOH 1027 16.329 18.885 28.375 1.00 19.76 ATOM 1023 O1 HOH1028 18.501 −6.259 40.698 1.00 14.77 ATOM 1024 O1 HOH 1029 18.291−13.454 23.318 1.00 15.78 ATOM 1025 O1 HOH 1030 10.174 5.278 12.637 1.0013.08 ATOM 1026 O1 HOH 1031 1.243 0.722 34.708 1.00 17.19 ATOM 1027 O1HOH 1032 2.523 −3.821 18.445 1.00 16.48 ATOM 1028 O1 HOH 1033 15.902−4.895 19.077 1.00 10.14 ATOM 1029 O1 HOH 1034 16.085 18.258 38.411 1.0020.04 ATOM 1030 O1 HOH 1035 8.277 3.225 41.854 1.00 12.99 ATOM 1031 O1HOH 1036 16.439 13.001 35.214 1.00 16.54 ATOM 1032 O1 HOH 1037 28.1315.663 18.293 1.00 20.92 ATOM 1033 O1 HOH 1038 14.371 −11.433 40.776 1.0017.19 ATOM 1034 O1 HOH 1039 6.487 15.695 21.119 1.00 14.88 ATOM 1035 O1HOH 1040 2.321 −12.928 26.470 1.00 11.64 ATOM 1036 O1 HOH 1041 10.6935.971 40.782 1.00 17.64 ATOM 1037 O1 HOH 1042 12.532 16.814 29.258 1.0017.91 ATOM 1038 O1 HOH 1043 15.955 −13.066 32.485 1.00 14.81 ATOM 1039O1 HOH 1044 18.984 3.255 12.162 1.00 21.70 ATOM 1040 O1 HOH 1045 10.02510.429 37.944 1.00 16.17 ATOM 1041 O1 HOH 1046 10.284 −14.324 27.9161.00 18.92 ATOM 1042 O1 HOH 1047 14.189 5.045 17.917 1.00 14.25 ATOM1043 O1 HOH 1048 1.288 −3.987 37.554 1.00 20.26 ATOM 1044 O1 HOH 104910.779 −7.111 14.686 1.00 24.75 ATOM 1045 O1 HOH 1050 11.650 13.76717.828 1.00 19.59 ATOM 1046 O1 HOH 1051 14.534 9.188 36.954 1.00 19.75ATOM 1047 O1 HOH 1052 19.606 −7.904 38.613 1.00 20.20 ATOM 1048 O1 HOH1053 9.427 9.736 48.436 1.00 15.68 ATOM 1049 O1 HOH 1054 −2.792 −4.53826.019 1.00 26.55 ATOM 1050 O1 HOH 1055 −0.725 −9.213 26.754 1.00 18.89ATOM 1051 O1 HOH 1056 −0.278 6.480 29.432 1.00 23.19 ATOM 1052 O1 HOH1057 25.587 −11.455 24.178 1.00 14.80 ATOM 1053 O1 HOH 1058 16.90010.379 36.579 1.00 17.89 ATOM 1054 O1 HOH 1059 3.126 15.011 20.955 1.0013.94 ATOM 1055 O1 HOH 1060 24.968 5.423 18.006 1.00 15.86 ATOM 1056 O1HOH 1061 18.376 −0.133 12.322 1.00 22.38 ATOM 1057 O1 HOH 1062 15.661−10.620 22.135 1.00 20.09 ATOM 1058 O1 HOH 1063 14.444 −8.692 41.5101.00 17.78 ATOM 1059 O1 HOH 1064 −2.099 2.685 19.269 1.00 18.56 ATOM1060 O1 HOH 1065 15.065 −14.513 30.168 1.00 20.78 ATOM 1061 O1 HOH 1066−0.478 −12.428 26.525 1.00 19.85 ATOM 1062 O1 HOH 1067 24.619 −2.27441.474 1.00 20.99 ATOM 1063 O1 HOH 1068 14.937 4.314 41.154 1.00 23.31ATOM 1064 O1 HOH 1069 18.536 3.179 40.506 1.00 20.39 ATOM 1065 O1 HOH1070 12.859 4.828 42.895 1.00 23.36 ATOM 1066 O1 HOH 1071 21.098 −7.93036.367 1.00 22.36 ATOM 1067 O1 HOH 1072 −4.171 −0.822 29.589 1.00 31.08ATOM 1068 O1 HOH 1073 5.760 16.825 18.804 1.00 26.85 ATOM 1069 O1 HOH1074 14.347 16.052 36.421 1.00 26.89 ATOM 1070 O1 HOH 1075 26.493 −4.49720.667 1.00 19.77 ATOM 1071 O1 HOH 1076 2.436 9.335 39.933 1.00 22.58ATOM 1072 O1 HOH 1077 28.166 −2.047 20.549 1.00 17.57 ATOM 1073 O1 HOH1078 26.366 −10.689 21.612 1.00 24.57 ATOM 1074 O1 HOH 1079 24.829−14.257 24.782 1.00 24.91 ATOM 1075 O1 HOH 1080 13.266 −13.588 42.0521.00 24.33 ATOM 1076 O1 HOH 1081 24.443 2.764 17.515 1.00 24.61 ATOM1077 O1 HOH 1082 4.269 −9.664 30.981 1.00 24.44 ATOM 1078 O1 HOH 10834.143 −10.890 22.698 1.00 22.21 ATOM 1079 O1 HOH 1084 18.040 4.74942.785 1.00 25.14 ATOM 1080 O1 HOH 1085 1.136 −9.675 22.102 1.00 27.39ATOM 1081 O1 HOH 1086 9.192 14.318 28.336 1.00 22.60 ATOM 1082 O1 HOH1087 19.935 −4.720 19.071 1.00 29.60 ATOM 1083 O1 HOH 1088 4.049 −5.33416.898 1.00 21.65 ATOM 1084 O1 HOH 1089 27.148 9.654 24.125 1.00 24.37ATOM 1085 O1 HOH 1090 16.984 11.029 13.815 1.00 27.26 ATOM 1086 O1 HOH1091 20.544 −2.126 18.367 1.00 22.99 ATOM 1087 O1 HOH 1092 23.707 3.15139.432 1.00 28.88 ATOM 1088 O1 HOH 1093 −1.920 −4.147 30.571 1.00 27.39ATOM 1089 O1 HOH 1094 11.899 −14.917 37.385 1.00 32.81 ATOM 1090 O1 HOH1095 20.795 14.376 16.474 1.00 29.46 ATOM 1091 S SO4 300 17.194 −14.45126.775 1.00 23.76 ATOM 1092 O1 SO4 300 17.779 −15.800 26.674 1.00 25.07ATOM 1093 O2 SO4 300 16.796 −13.993 25.435 1.00 24.21 ATOM 1094 O3 SO4300 16.011 −14.509 27.652 1.00 24.52 ATOM 1095 O4 SO4 300 18.190 −13.52427.343 1.00 23.52 ATOM 1096 S SO4 301 13.826 −5.555 16.116 1.00 19.81ATOM 1097 O1 SO4 301 13.393 −6.524 15.095 1.00 21.58 ATOM 1098 O2 SO4301 13.720 −4.187 15.584 1.00 19.29 ATOM 1099 O3 SO4 301 12.996 −5.69317.324 1.00 20.41 ATOM 1100 O4 SO4 301 15.233 −5.831 16.467 1.00 22.09ATOM 1101 S SO4 302 13.249 23.091 32.541 1.00 76.58 ATOM 1102 O1 SO4 30213.950 22.101 31.704 1.00 76.66 ATOM 1103 O2 SO4 302 12.256 22.40933.391 1.00 76.74 ATOM 1104 O3 SO4 302 14.229 23.787 33.395 1.00 76.89ATOM 1105 O4 SO4 302 12.559 24.071 31.680 1.00 76.61 END

1. An isolated Fibroblast Growth Factor 4 (FGF4) antagonist polypeptidehaving the amino acid sequence set forth for FGF4 in FIG. 2 (top line)(SEQ ID NO: 20) with substitution of an alanine amino acid residue for:(a) threonine at amino acid residue
 196. 2. A mutant Human FibroblastGrowth Factor 4 (FGF4) antagonist polypeptide having the amino acidsequence set forth in SEQ ID NO. 20, wherein at least one amino acidresidue in the secondary binding site selected from Leu 162, Pro 195 andThr 196 is substituted with alanine.